Human p51 genes and gene products thereof

ABSTRACT

Novel human genes falling within the category of family genes relating to p53 gene which is known as a cell proliferation regulatory gene, and gene products thereof. A human p51 gene characterized by containing a base sequence encoding an amino acid sequence represented by SEQ ID NO:1; a human p51 gene having a base sequence consisting of the 145- to 1488-bases in the sequence represented by SEQ ID NO:2; vectors containing these genes; host cells transformed with these vectors; a process for producing a p51 protein having the amino sequence represented by SEQ ID NO:1; which comprises culturing the above host cells and harvesting the protein from the thus obtained culture; and the p51 protein having the amino acid sequence represented by SEQ ID NO:1.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.09/670,568, filed Jan. 18, 2001 now U.S. Pat. No. 7,132,276; which is a371 of PCT/JP99/01512, filed Mar. 24, 1999; the disclosure of each ofwhich is incorporated hereby in reference.

TECHNICAL FIELD

The present invention relates to novel human genes. More particularly,the invention relates to a novel human gene analogous to human p53 andhuman p73 genes, which are known as tumor suppressor genes, and thecorresponding gene product.

BACKGROUND ART

The p53 protein was discovered as a nuclear protein binding to the largeT antigen of the DNA tumor virus SV40 and its gene (p53 gene) has beencloned. At first, the p53 gene was considered to be an oncogene becausethe transfer of this gene and the ras gene together into cells resultedin transformation of embryonal cells. Later studies, however, revealedthat the initially cloned p53 gene was a mutant type and that the wildtype rather suppressed the transforming activity of the mutant type. Bynow, deletions or anomalies in the p53 gene have been detected in manyhuman cancers and a gamate mutation of the p53 gene was also discoveredin Li-Fraumeni syndrome which is known to be a hereditary disease with ahigh risk for malignant conversion. Because of these and other findings,the p53 gene has by now been considered to be an important suppressoroncogene [Baker, S. J., et al., Science, 244, 217-221 (1989): Nigro, J.M., Nature, 342, 705-708 (1989)].

The human p53 protein consists of 393 amino acid residues and can beroughly divided into the N-terminal domain (the 1˜101 st amino acidregion), the core domain (the 102˜292 nd amino acid region), and theC-terminal domain (the 293˜393 rd amino acid region). The N-terminaldomain contains sequences necessary for transcriptional regulation, suchas acidic amino acids and a high-proline region, and is considered to bea transcriptional activator domain. The central core domain contains 3hydrophobic sites and is a domain associated with nucleotidesequence-specific DNA binding. The C-terminal domain contains many basicamino acids and a sequence necessary for tetramerization and isconsidered to be responsible for recognition of nonspecific DNA bindingand DNA damage and inhibition of transformation.

Many of the p53 gene abnormalities detected in human cancer cells aremissense mutations and most of them are concentrated in the core domaincorresponding to the 100˜300 th amino acid sequence from the N terminus,particularly in the region called “hot-spot” which has been conservedamong species. The hot-spot region in the core domain is the sequenceassociated with the binding between p53 protein and DNA and, actually,mutation of this region results in the inhibition of specific binding toDNA.

It became clear from the above that the p53 protein plays the role of atranscriptional control factor which binds specifically to other genesto modulate expression of the genes.

The gene whose transcription is induced by the p53 protein includes,among others, the p21 gene [known as WAF1, CIP1, or SDI1 (EI-Dairy, W.S., et al., Cell, 75, 817 (1993)); MDM2 (Wu. X., et al., Genes Dev., 7,1126 (1993)); MCK (Weintraub. H., et al., Proc. Natl. Acad. Sci. USA,88, 4570 (1991): Zambetti. G. P., et al., Genes Dev., 6, 1143 (1992))],GADD45 [Kastan, M. B., et al., Cell, 71, 587 (1992)], Cyclin G [CyclinG: Okamoto, K., EMBO J., 13, 4816 (1994)], BAX [Miyashita, T., et al.,Cell, 80, 293 (1995)], and insulin-like growth factor-binding protein 3[IGF-BP3: Buckbinder, L., et al., Nature, 377 646 (1995)].

The protein encoded by the p21 gene is an inhibitor protein forcyclin-dependent kinase (CDK), and it has been found that the wild typep53 protein regulates the cell cycle in an inhibitory way through p21[Harper, J. W., et al., Cell, 75, 805 (1993): Xiong, Y., et al., Nature,366, 707 (1993): Gu, Y., et al., Nature, 366, 701 (1993)]. Furthermore,the p21 gene reportedly binds to the proliferating cell nuclear antigen(PCNA) to directly inhibit DNA replication [Waga, S., et al., Nature,369, 574 (1994)]. In addition, the p21 gene has been found to the samegene as the SDI1 gene which induces senescence of cells to inhibit DNAsynthesis [Noda., A., et al., Exp. Cell Res., 211, 90 (1994)].

MDM2 binds to the p53 protein to inactivate the transcriptionalregulation activity of the gene protein, leading to the putativeconclusion that MDM2 is acting as a negative feedback regulating factor.

IGF-BP3 is a negative regulating factor in IGF signalization. Therefore,the increase of the IGF-BP3 gene by the p53 protein suggests thepossible outcome that the p53 protein induces suppression of growth ofIGF-dependent cells.

Meanwhile, the wild type p53 protein reportedly induces apoptosis ofmyelocytic leukemia cells [Yonish-Rouach, E., et al., Nature, 352, 345(1991)]. Induction of thymocyte apoptosis by irradiation does not takeplace in p53-defective mice [Lowe, S. W., Nature, 362, 847 (1993):Clarke, A. R., et al., Nature 362, 849 (1993)] and, in the crystallinelens, retina and brain, the p53 protein induces apoptic death of cellsdeprived of normal retinal blastoma gene (RB gene) activity [Pan, H.,and Griep, A. E., Genes Dev., 8, 1285 (1994): Morgenbesser, S. D., etal., Nature 371, 72 (1994): Howes, K. A., Genes Dev., 8, 1300 (1994):Symonds, H., et al., Cell, 78, 703 (1994)]. E. White proposes that thep53 protein is useful for a surveillance of RB gene mutation and thatthe protein is likely to induce apoptosis of the cells in which a RBgene mutation is involved [White, E., Nature, 371, 21 (1994)].

Furthermore, in the mouse erythroid leukemia cell line in which thetemperature-sensitive p53 gene only is expressed, a fall in temperatureresults in reconversion of the mutant p53 gene to the wild type toinduce apoptosis and the mutant p53 gene isolated therefrom imparts theability to grow in soft agar medium to a p53-defective fibroblast line(impart anchorage independence) [Xu et al., Jpn, J. Cancer Res. 86:284-291 (1995); Kato et al., Int. J. Oncol. 9: 269-277].

BAX is able to bind to bc1-2, which is an inhibitor of apoptosis, andencouratges apoptic cell death [Oltvai, Z. M., et al., Cell, 74, 609(1993)]. The increase in the BAX gene and decrease in bc1-2 by the p53protein are involved in the apoptosis of the mouse leukemia cell line M1[Miyashita, T., et al., Oncogene, 9, 1799 (1994)] and Fas, which is oneof the signal transducers for apoptosis, is increased in non-small-celllung cancer and erythroleukemia [Owen-Schaub, L. B., et al., Mol. CellBiol., 15, 3032 (1995)].

The many investigations referred to above have revealed that the p53protein either activates or represses the transcription of various genesnot limited to the p21 gene. Moreover, even the mutant p53 proteindefected in the transcriptional regulating function is capable ofinteracting with other intracellular proteins to transmit signals anddischarge a DNA damage repairing function.

Among the functions of the p53 protein which have so far been identifiedare a transcription regulating function, a signal transducer functionthrough binding to other intracellular proteins, a constituent elementof a protein complex related to DNA replication, a DNA binding function,and exonuclease activity, and it is conjectured to be the result of acompound interplay of these functions that causes the arrest of the cellcycle in cells, induction of apoptosis, DNA repair, regulation of DNAreplication, and induction of differentiation.

Furthermore, it is not true that the functions of the p53 protein areexpressed only in the event of a gene damage but it is reported thatwhen the living tissue is subjected to various stresses such as viralinfection, cytokine stimulation, hypoxia, a change in the nucleotidepool, drug-induced metabolic abnormality, etc., the stimuli triggerquantitative or qualitative changes in the p53 protein. The p53 proteinsubjected to the quantitative or qualitative regulation expresses itsfunctions, such as signal transduction through interactions with otherproteins and control of the transcription of other genes, to regulatethe replication of DNA in cells of the living tissue subjected tobiological stresses, repair the cells by suspending the cell cycle,eliminate cells by way of apoptosis, or promote the differentiation ofcells, thereby contributing to the protection of the living tissueagainst the stresses [Ganman, C. E., et al., Genes Dev., 9, 600-611(1995): Graeber, T. G., et al., Nature, 379, 88-91 (1996): Linke, S. P.,et al., Genes Dev., 10, 934-947 (1996): Xiang, H., et al., J. Neurosci.,16, 6753-6765 (1996)].

In view of the existence of p53 gene mutations in a half of humantumors, clinical application of the p53 gene and its product protein tothe diagnosis and therapy of tumors has been a subject of study inrecent years. The method of detecting tumor cells invading the lymphnode or body fluid by carrying out a PCR using primers specificallyrecognizing the mutation site of the p53 gene can be an effectivediagnostic technique for estimating the scope of tumor invasion orpredicting a recurrence of the tumor [Hayashi, H., et al., Lancet, 345,1257-1259 (1995)].

Furthermore, taking advantage of the apoptosis-inducing activity of thep53 protein, a gene therapy comprising introducing a wild type p53 geneinto the tumor cell by means of a virus vector is being practiced in theUnited States and its effectiveness has been reported [Roth, J. A., etal., Nature Med., 2, 985-991 (1996)]. Recently, in Japan, too, this genetherapy has been started in several locations.

Meanwhile, more than the majority of human tumors are not associatedwith p53 gene mutation and, from this fact, the possibility of existenceof other tumorigenesis-inhibitory proteins analogous to the p53 proteinhas been pointed out.

The inventors of the present invention previously found that a p53 genemutation cannot be a useful premonitory indicator of non-Hodgkin'slymphoma (NHL).

Recently, a novel gene, named p73, which has high homology to said p53gene has been identified [Kaghad, M., et al., Cell, 90, 809-819 (1997)].According to the information available to the present inventors, the p73protein shows 29% homology to the human p53 protein in thetranscriptional activator domain (the 1 st˜45 th amino acid region).Moreover, this p73 protein has a homology of 63% in the DNA bindingdomain (the 113 rd˜290 th amino acid region) having 6 complementaryconserved sequences called hot spots of mutation; and a homology of 38%in the oligomerization domain (the 319 th˜363 rd amino acid region).With regard to the C-terminal domain, however, no significant homologyhas been recognized between p73 protein and p53 protein.

It is reported that excessive expression of the p73 protein inhibits thegrowth of a neuroblastoma cell line and SAOS2 cells (an osteosarcomacell line) and that a transient expression of the p73 protein promotesthe apoptosis of SAOS2 cells and baby hamster's kidney cells [BruceClurman and Mark Groudine, Nature, 389, 122-123 (1997): Christine, A.,et al., Nature, 389, 191-194 (1997)].

However, the p73 protein is somewhat different from the p53 protein inthat the former is expressed only at low levels in normal tissues.Moreover, the p73 protein is different from the p53 protein in that theexpression of the former protein in a neuroblastoma cell line is notinduced by UV irradiation or a low dose of actinomycin D.

Therefore, it is not true that the p73 protein has the exactly the samefunctions as those of the p53 protein and, at the present, much dependson further research. There is a report arguing that, based on theobservations so far made, this p73 may be categorized as a putativetumor suppressive factor in neuroblastoma.

The present invention has for its object to provide information on anovel gene and gene product related to the morphogenesis of humantumors. More particularly, the object of the present invention is toprovide a novel gene analogous to the p53 gene which, as mentionedabove, is already known as a tumor suppressor gene and the correspondinggene product.

It is a further object of the present invention to provide primers andprobes each comprising a partial DNA of said gene, vectors harboringsaid gene, transformants as transformed using any of said vectors, and amethod of producing said gene product which comprises growing any ofsaid transformants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating features of the structural domains ofthe p51A protein, along with those of the p53 protein and p73β protein.In the diagram, “TA” represents a transcriptional activator domain; “DNAbinding” represents a DNA binding domain; and “Oligo” represents anoligomerization domain.

FIG. 2 is a diagram showing the homology relationship of the amino acidsequence encoded by the human p51A gene (SEQ ID NO:1), the amino acidsequence of the p53 protein (SEQ ID NO:3), and the amino acid sequenceof the p73β protein (SEQ ID NO:6). The amino acids which are commonamong the three sequences are indicated in blocks. The consensussequence is SEQ ID NO:7.

FIG. 3 is a diagram showing the homology relationship of the amino acidsequence encoded by the human p51B gene (SEQ ID NO:4) and the amino acidsequence of the p73α protein (SEQ ID NO:8). The amino acids which arecommon to both sequences are indicated in blocks. The consensus sequenceis SEQ ID NO:9.

FIG. 4 is a schematic diagram comparing the structure of the alternativesplicing variant (p51A, p51B) of the p51 protein with the structure ofthe alternative splicing variant (p73α, p73β) of the p73 protein.

FIG. 5 is a photograph, in lieu of a drawing, which shows the pattern ofexpression of p51mRNA in various human tissues as a Northern blotting(using a Clonetech's filter) electrophoretogram. The lanes represent theresults for 1: heart, 2: brain, 3: placenta, 4: lung, 5: liver, 6:skeletal muscle, 7: spleen, 8: pancreas, respectively.

FIG. 6 is a photograph, in lieu of a drawing, which shows the pattern ofexpression of p51mRNA in various human tissues as a Northern blotting(using a filter prepared by using the RNA purchased from Clontech)electrophoretogram. The lanes represent the results for 1: mammarygland, 2: prostate, 3: salivary gland, 4: stomach, 5: thymus, 6:thyroid, 7: trachea, and 8: uterus, respectively.

FIG. 7 is a photograph, in lieu of a drawing, which shows theanti-colony forming activity of the p51A gene. More specifically, it isa photograph, in lieu of a drawing, which shows in comparison the colonyforming activities of the cells transformed with the p51A expressionplasmid (p51A), p53 expression plasmid (p53), HA-tagged p51A expressionplasmid (HAp51A), and vector (RcCMV) alone, respectively.

FIG. 8 is a schematic diagram showing the reporter constructs used inExperimental Example 2. In the diagram, “WAF-1 promoter luc” representsa wild type p21WAF1 promoter construct with two p53 regulating elementsretained; “del 1” represents a similar construct in which one upstreamelement has been deleted; and “del 2” represents a construct in whichboth elements have been deleted.

FIG. 9 is a diagram showing the transactivation activity found in thetransfer of the p51A expression plasmid (p51A), p53 expression plasmid(p53) or control vector (Rc/CMV) harboring the various reporterconstructs shown in FIG. 8 into SAOS2 cells [cf Experimental Example 2].

FIG. 10 is a diagram showing the transactivation activity found in thetransfer of the p51A expression plasmid (p51A), HA-labeled p51Aexpression plasmid (HAp51A), p53 expression plasmid (p53) or controlvector (Rc/CMV) harboring the PGC reporter construct, whose p53 responsehas been experimentally demonstrated, into SAOS2 cells [cf ExperimentalExample 2].

FIG. 11 is a photograph (an ethidium bromide-stained agarose gelelectrophoretogram), in lieu of a drawing, which shows the results ofDNA fragmentation assays performed with 1C1 and 4B1 cells containing thehuman p51A gene and 1-2-3 cells not containing the p51A gene as grown atdifferent temperatures of 32° C. and 37° C.

In the diagram, the “1-2-3 cells” represents control cells into whichthe vector only has been introduced and not containing the p51A gene andthe “1C1 cells” or “4B1 cells” represents the p51A-containing 1-2-3cells as transformed with the expression vector harboring the p51A gene(pRcCMV/p51A). The “λ/Hind III” represents digestion products asdigested with the λ phage DNA restriction enzyme Hind III and are DNAsize markers (product of New England Biolabs. Ind.). The “100 bp ladder”represents size markers comprising DNA fragments having sizescorresponding to multiples of 100 bp (product of GIBCO-BRL).

FIGS. 12˜14 show a diagram comparing the nucleotide sequence (bottomrow) of the coding region of the human p51B gene (SEQ ID NO:5) with thecorresponding sequence (upper row) of the mouse homolog (mouse p51Bgene; SEQ ID NO:10)). The nucleotides common between the two sequencesare indicated by the asterisk mark in the diagram.

FIG. 15 is a diagram comparing the amino acid sequences of the humanp51B protein (SEQ ID NO:4) and mouse p51B protein (SEQ ID NO: 11)encoded by the human p51B gene and mouse p51B gene, both shown in FIGS.12˜14, respectively. The amino acids common to both sequences areindicated by the asterisk mark in the diagram.

DISCLOSURE OF INVENTION

Since more than the majority of human tumor tissues have multants of thep53 gene which is an oncogene suppressor gene as mentioned above, thepossibility has been suggested of the existence of other gene products(proteins) than the p53 protein which are discharging thetumorigenesis-inhibiting function.

Therefore, the inventors of the present invention did intensiveinvestigations in search for novel genes and gene products which mightbe associated with said tumorigenesis-inhibiting function. As a result,they discovered a novel human-derived gene coding for a protein showingactivity similar to that of said p53 protein and confirmed that theparticular gene or gene product is significantly associated withapoptosis. The present invention has its basis in this new finding.

The present invention, therefore, is directed to the following human p51genes 1˜8 and the related genes.

1. A gene coding for the following protein (a) or (b):

-   (a) a protein having the amino acid sequence shown under SEQ ID NO:1-   (b) a protein having an amino acid sequence derived from the amino    acid sequence shown under SEQ ID NO: 1 by deletion, substitution or    addition of one or a plurality of amino acids and having p51    activity.

2. A gene comprising the following DNA (a) or (b):

-   (a) a DNA having a nucleotide sequence identified by the nucleotide    numbers 145˜1488 of the nucleotide sequence shown under SEQ ID NO:2-   (b) a DNA capable of hybridizing with the DNA having a nucleotide    sequence identified by the nucleotide numbers 145˜1488 of the    nucleotide sequence shown under SEQ ID NO:2 under stringent    conditions and coding for a protein having p51 activity.

3. A gene as defined in paragraph 2 which has the nucleotide sequenceshown under SEQ ID NO:2.

4. A cDNA comprising the following DNA (a) or (B):

-   (a) a DNA having a nucleotide sequence identified by the nucleotide    numbers 145˜1488 of the nucleotide sequence shown under SEQ ID NO:2-   (b) a DNA capable of hybridizing with a DNA having a nucleotide    sequence identified by the nucleotide numbers 145˜1488 of the    nucleotide sequence of SEQ ID NO:2 under stringent conditions and    coding for a protein having p51 activity.

5. A DNA characterized in that it is capable of hybridizing with thenucleotide sequence of SEQ ID NO:2 under stringent conditions.

6. A DNA characterized in that it is capable of the hybridizing with anucleotide sequence identified by the nucleotide numbers 145˜1488 of SEQID NO:2 under stringent conditions.

7. The DNA defined in paragraph 5 for use as a primer.

8. The DNA defined in paragraph 5 for use as a probe.

The present invention is further directed to the following human p51proteins 9˜14 and the related proteins or peptides.

9. A protein defined under (a) or (b) below:

-   (a) a protein having the amino acid sequence shown under SEQ ID NO:1-   (b) a protein having an amino acid sequence derived from the amino    acid sequence of SEQ ID NO:1 by deletion, substitution or addition    of one or a plurality of amino acids and having p51 activity.

10. A protein as defined in paragraph 9 at least containing the aminoacid sequences identified by the amino acid numbers 1˜59, amino acidnumbers 142˜321, and amino acid numbers 359˜397 of the amino acidsequence shown under SEQ ID NO:1.

11. A polypeptide having an amino acid sequence, in SEQ ID NO:1, whichhas at least one function selected from the group consisting oftranscriptional activation function, DNA binding function andoligomerization function.

12. A polypeptide as defined under (a) or (b) below:

-   (a) a polypeptide having an amino acid sequence identified by the    amino acid numbers 1˜59 of SEQ ID NO:1-   (b) a polypeptide having an amino acid sequence derived from the    amino acid sequence defined under (a) by deletion, substitution or    addition of one or a plurality of amino acids and having a    transcriptional activation function.

13. A polypeptide as defined under (a) or (b) below:

-   (a) a polypeptide having an amino acid sequence identified by the    amino acid numbers 142˜321 of SEQ ID NO:1-   (b) a polypeptide having an amino acid sequence derived from the    amino acid sequence defined under (a) by deletion, substitution or    addition of one or a plurality of amino acids and having a DNA    binding function.

14. A polypeptide as defined under (a) or (b) below:

-   (a) a polypeptide having an amino acid sequence identified by the    amino acid numbers 359˜397 of SEQ ID NO:1-   (b) a polypeptide having an amino acid sequence derived from the    amino acid sequence defined under (a) by deletion, substitution or    addition of one or a plurality of amino acids and having an    oligomerization function.

The present invention is further directed to a vector harboring said p51gene, host cells transformed with said vector, and a method of producingthe p51 protein characterized by growing said host cells in a medium andharvesting the protein from the resulting culture.

It should be understood that the designation of p51 is used only forconvenience's sake in this specification and is by no means definitiveof the gene and gene product (protein) of the present invention.

Furthermore, the term “gene (DNA)” in the context of the presentinvention means not only a double-stranded DNA but also asingle-stranded DNA, inclusive of the component sense chain andantisense chain, and is by no means limitative of its length. Therefore,unless otherwise indicated, the gene (DNA) of the present inventionincludes the double-stranded DNA inclusive of human genomic DNA, asingle-stranded DNA comprising the cDNA (sense chain), a single-strandedDNA having a sequence complementary to said sense chain (antisensechain), and all fragments thereof.

The representation of amino acids, peptides, nucleotide sequences,nucleic acids, etc. by abbreviations in this specification confirms tothe recommendations by IUPAC-IUB, the “Guidelines for Preparation ofSpecifications Etc. which contain Nucleotide Sequences or Amino AcidSequences” (the Japanese, United States of America and European TrinityPatent Office), and the conventions in the use of symbols in the relatedfield of art.

(1) The p51 Gene and its Equivalent

The present invention relates to novel human genes coding for proteinshaving actions or functions identical or equivalent to the actions orfunctions of the p53 protein.

The gene according to the present invention has been acquired bycarrying out a PCR using primers newly established after energeticexplorations using specific regions selected from the sequences of thehitherto-known p53 gene and p73 gene with judicious endeavors.

More particularly, by carrying out a PCR using the novel primersdescribed in Examples which appear hereinafter, a gene fragment which isnot identical but similar to the p53 gene and p73 gene was obtained. Byusing this DNA fragment as a probe, a cDNA clone coding for a novelprotein having high homology to the amino acid sequence of p53 proteinwas successfully isolated from among cDNA clones randomly selected froma human skeletal muscle cDNA library.

The calculated molecular mass of the amino acid sequence deducted fromthe cDNA thus obtained was about 50,894 Da and, therefore, the presentinventors named this cDNA (DNA) “human p51A gene (or briefly, p51Agene)” and the protein having the amino acid sequence encoded by thisgene “p51A protein (or briefly, p51A protein)” for convenience's sake.

Subsequent research revealed that the gene encoded by the p51 cDNA clonehas alternative splicing variants. Moreover, an investigation of thepattern of expression and production of the transcripts of the gene invarious human tissues revealed that the expression products (proteins)exist as spliced chiefly in a short form and a long form.

Based on the amino acid information deduced from the p51 cDNA splicingvariants, the shot-form splicing variant is the gene (p51A gene) codingfor the protein having said 448 amino acid sequence (molecular mass ca50.9 kDa) and the long-form splicing variant is a gene coding for aprotein having a 641 amino acid sequence (molecular mass ca 71.9 kDa).In this specification, for convenience's sake, the latter gene is called“human p51B gene (or briefly, p51B gene)” and the protein having theamino acid sequence encoded by said gene “human p51B protein (orbriefly, p51B protein)”.

Furthermore, in this specification, said p51A gene and p51B gene arecollectively referred to as “p51 gene” and the p51A protein and p51Bprotein are collectively referred to as “p51 protein”.

Referring to said splicing variant of the p51 gene, the existence of aplurality of variants inclusive of the gene defective of a part of theTA domain have been confirmed.

An investigation of the expression products of these p51 genes revealedthat the p51 gene product (p51 protein) of the present invention showstranscriptional activation activity, cell growth-inhibitory activity andapoptosis-inducing activity which are similar to the activities of thep53 protein. Furthermore, the expression of the p51 gene in humantissues was found to be more tissue-specific than the expression of thep53 gene and, compared with the expression of the p73 gene which is alsotissue-specific, was broader in tissue distribution although there wasan overlap of expression pattern between them. Moreover, the mutation ofthe p51 gene was found in the human tumor tissues or tumor cell lines.

The above findings suggested strongly that the human p51 gene of thepresent invention is a new member of the p53 tumor suppressor genefamily.

As a specific example of the p51 gene according to the presentinvention, there can be mentioned one having the DNA sequence possessedby the clones (p51A, p51B) described in Example 1 which appearshereinafter.

As the gene possessed by the p51A clone, there can be mentioned the gene(1344 nucleotides) coding for the 448-residue protein of SEQ ID NO:1 inthe SEQUENCE LISTING which appears hereinafter. Specifically, this is agene having the nucleotide sequence corresponding to the 145 th˜1488 thnucleotides of SEQ ID NO:2, which corresponds to an open reading frame.

The full-length nucleotide sequence of the p51A cDNA consists of 2816nucleotides as shown in SEQ ID NO:2. The p51A gene of the presentinvention includes genes containing this nucleotide sequence shown underSEQ ID NO:2. In the nucleotide sequence shown under SEQ ID NO:2, theinitiation codon (ATG) is situated in the 145˜147 nucleotide positionand the polyadenylation signal (AATAA) is situated in the 2786˜2791position.

The amino acid sequence of the 448-residue p51A protein encoded by thep51A gene is shown under SEQ ID NO:1. As shown, this protein has atranscriptional activation domain corresponding to the amino acidnumbers 1˜59, a DNA binding domain corresponding to the amino acidnumbers 142˜321, and an oligomerization domain corresponding to theamino acid numbers 353˜397.

The homology of each of said domains of the p51A protein to thecorresponding domain of the known proteins p53 or p73 was investigatedwith FASTA Program using the GCG software (Wisconsin Sequencing Package,Genetics Computer Group) [Person, W. R. and Lipman, D. J., Proc. Natl.Acad. Sci. U.S.A., 85, 1435-1441 (1988)]. The results are shown in Table1 (cf. FIGS. 1 and 2). For reference, the homologies between p53 proteinand p73β protein as determined by the same method are also shown inTable 1.

TABLE 1 Transcription Full-length activation DNA binding Oligomeriza-sequence domain domain tion domain p51A

p53 36% 22% 60% 37% p51A

P73β 42% 30% 87% 65% p53

p73 28% 27% 63% 83%

On the other hand, as the gene possessed by the p51B clone, there can bementioned a gene (1923 nucleotides) coding for the 641-residue proteinshown under SEQ ID NO:4 in the SEQUENCE LISTING given hereinafter.Specifically, this is a gene having the nucleotide sequence identifiedby the nucleotide numbers 145˜2067 of SEQ ID NO:5, which corresponds toan open reading frame.

The full-length nucleotide sequence of this p51B cDNA consists of 2270nucleotides as shown under SEQ ID NO:5. The p51B gene according to thepresent invention includes genes containing the nucleotide sequenceshown under SEQ ID NO:5.

The amino acid sequence of the 641-residue p51B protein encoded by thep51B gene is shown under SEQ ID NO:4. This protein has a transcriptionalactivation domain corresponding to the amino acid numbers 1˜59, a DNAbinding domain corresponding to the amino acid numbers 142˜321, and anoligomerization domain corresponding to the amino acid numbers 353˜397.In addition, there is an additional sequence (SAM domain) in theC-terminal region, although the corresponding amino acid numbers cannotbe identified. In this specification, the region of amino acid numbers353˜641 inclusive of this SAM domain is regarded as an oligomerizationdomain in a broad sense.

As in the case of the p51A protein, the homology of the amino acidsequence of each of said domains of the p51B protein to the sequence ofthe corresponding domain of the known protein p73α was investigated withFASTA PROGRAM using GCG software. The results are shown in FIG. 3. InFIG. 3, the boxed parts are amino acid sequences common to the p51Bprotein and p73α protein. It is, therefore, clear that the amino acidsequence of the p51B protein according to the present invention ishomologous to the sequence of the p73α protein over a broad range.

Thus, the p51 gene according to the present invention includes a humanp51A gene having a nucleotide sequence coding for a protein having theamino acid sequence shown under SEQ ID NO:1 and a human p51B gene havinga nucleotide sequence coding for a protein having the amino acidsequence shown under SEQ ID NO:4. However, the p51 gene of the inventionis not limited to those genes but includes all homologs of those humanp51 genes.

The term “homolog of human p51 gene” means any member of a group ofinterrelated genes which are analogous to said p51A gene or p51B gene inthe nucleotide sequence and/or structural features and gene expressionpattern or analogous to each other in the biological functions of theirown and gene products (proteins) and, as such, may be regarded asconstituting one gene family. In this sense, splicing variants andalleles of the human p51 gene are, of course, subsumed in the concept ofsaid “homolog”.

As an example of said homolog, there can be mentioned a gene which codesfor a protein having an amino acid sequence resulting from the mutationor modification of one or a plurality of sites of the amino acidsequence shown under SEQ ID NO:1 and having actions or functions similarto those of said p51A protein having the amino acid sequence shown underSEQ ID NO:1. The preferred is a gene coding for an amino acid sequenceretaining at least a given degree of homology to the amino acid sequenceshown under SEQ ID NO:1.

The degree of homology in amino acid sequence may generally be not lessthan about 45%, preferably not less than about 50%, in terms of thefull-length sequence as determined with FASTA PROGRAM using said GCGsoftware. Preferably, the homology should be not less than a given valuein at least one of the transcriptional activation domain, DNA bindingdomain and oligomerization domain. For example, the homology in thetranscriptional activation domain may be about 35% or higher, preferablynot less than 45%, the homology in the DNA binding domain may be 88% orhigher, preferably not less than about 90%, and the homology in theoligomerization domain may be about 70% or higher, preferably not lessthan about 80%.

Thus, the gene of the present invention includes any gene having anucleotide sequence coding for a protein having an amino acid sequencederived from the sequence of SEQ ID NO:1 by deletion, substitution oraddition of one˜a few or a plurality of amino acids on condition thatthe above-mentioned qualifications are satisfied.

The extent of “deletion, substitution or addition of amino acids” andthe site or sites involved are not particularly restricted inasmuch asthe protein so modified is functionally equivalent to the protein (p51Aprotein or p51B protein) having the amino acid sequence of SEQ ID NO:1or 4. Thus, the term “p51 activity” as used in this specification meansthe activities and functions possessed by the p51 protein, representedby p51A protein or p51B protein, of the present invention, thusincluding tumor cell growth inhibitory activity, apoptosis-inducingactivity and transcriptional regulation function in cells, among others.

The p51 protein of the present invention is considered to have actionssimilar to those of the p53 protein which is known to be a cellproliferation inhibitory factor. Therefore, the term “p51 activity” asused in this specification referring to the actions and functions of thep51 protein may be restated in the same terms as applied to the knownactivities and functional features of the p53 protein.

As the actions and functions of the p53 protein, there may be mentioneda transcriptional regulation function, a signal transduction functionwhich is expressed through its binding to other intracellular proteins,the function as a component of the protein complex related to DNAreplication, a DNA binding function, exonuclease activity, etc., and asthe functions expressed by the composite interplay of said variousfunctions, a cell cycle interrupting function, an apoptosis-inducingfunction, a DNA repairing function, a DNA replication control functionand/or a differentiation-inducing function in cells. It is consideredthat the p51 protein of the present invention has some or all of theseactions and functions.

The modification of an amino acid sequence may be spontaneous, e.g.spontaneous mutation or posttranslational modification, but can beartificially induced on the basis of a native gene.

The present invention encompasses all modified genes coding for proteinshaving the above-mentioned characteristics of the p51 protein of theinvention without regard to the cause or means of mutation ormodification.

The means for making such artificial modifications includes geneticengineering techniques such as site-specific (-directed) mutagenesis[Methods in Enzymology, 154: 350, 367-382 (1987); ditto 100: 468 (1983);Nucleic Acids Res., 12: 9441 (1984); Zoku Seikagaku Jikken Koza 1“Idenshi Kenkyuho II” [Experimental Biochemistry Series 1 “Methods forGene Research II” (edited by Japanese Biochemical Society), p105(1986)], etc. and chemical synthetic techniques such as thephosphotriester method and the phosphoamidate method [J. Am. Chem. Soc.,89: 4801 (1967); ditto 91: 3350 (1968); Science, 150: 178 (1968);Tetrahedron Lett., 22: 1859 (1981); ditto 24: 245 (1983)] as well as asuitable combination of such techniques. More specifically, DNAsynthesis can be carried out chemically by the phosphoramidide method orthe triester method, or on a commercial automatic oligonucleotidesynthesizer. The double-stranded chain fragment can be obtained bysynthesizing complementary chains and annealing them together undersuitable conditions or can be obtained from a chemically synthesizedsingle-stranded chain by adding a complementary chain using a DNApolymerase together with suitable primer sequences.

As specific examples of the gene of the invention, there can bementioned the gene having a nucleotide sequence corresponding to thenucleotide numbers 145˜1488 of the nucleotide sequence shown under SEQID NO:2 and the gene having a nucleotide sequence corresponding to thenucleotide numbers 145˜2067 of the sequence of SEQ ID NO:5. Each ofthese nucleotide sequences represents an example of combination of thecodons coding for the respective amino acid residues of the amino acidsequence shown under SEQ ID NO:1 or 4. Therefore, the gene of thepresent invention is not limited to genes having such specificnucleotide sequences but may have nucleotide sequences designed by usinga combination of optional codons for each amino acid residue. Selectionof codons can be made in the routine manner, for example with referenceto the frequency of utilization of each codon by the host to be used[Nucleic Acids Res., 9, 43 (1981)].

Furthermore, as mentioned above, the gene of the present inventionincludes a nucleotide sequence having a defined degree of homology tothe nucleotide sequence corresponding to the nucleotide numbers 145˜1488[hereinafter sometimes referred to briefly as the nucleotide sequence(145-1488)] of the nucleotide sequence shown under SEQ ID NO:2.

As an example of such gene, there can be mentioned a gene having anucleotide sequence capable of hybridizing with a DNA having saidnucleotide sequence (145-1488) under stringent conditions, for examplein 0.1% SDS-containing 0.2×SSC at 50° C. or in 0.1% SDS-containing 1×SSCat 60° C.

The gene of the present invention can be easily produced and acquired bythe standard genetic engineering techniques [Molecular Cloning 2d Ed,Cold Spring Harbor Lab. Press (1989); Zoku Seikagaku Jikken Koza 1“Idenshi Kenkyuho I, II, III” [Supplemental Biochemical ExperimentalSeries 1 “Methods for Gene Research I, II, III” (edited by JapaneseBiochemical Society), (1986), etc.] based on the sequence information onthe specific examples shown in SEQ ID NO:2.

More particularly, the object gene can be acquired by constructing acDNA library from a suitable source in which the gene of the inventioncan be expressed and selecting the desired clone from this cDNA libraryusing a suitable probe or antibody specific to the gene of the inventionin the per se known manner [Proc. Natl. Acad. Sci., USA., 78: 6613(1981); Science, 222: 778 (1983), etc.].

In the above procedure, the cDNA source includes but is not limited tovarious cells or tissues in which the gene of the invention is expressedand cultured cells derived therefrom. Isolation of the whole RNA fromsuch a source, isolation and purification of mRNA, acquisition of cDNA,and cloning thereof can all be carried out in the routine manner. cDNAlibraries are also commercially available. In the practice of thepresent invention, such commercial cDNA libraries, for example thoseavailable from Clontech Lab. Inc., can also be employed.

The method of screening for the gene of the invention from a cDNAlibrary is not particularly restricted, either, but the conventionalmethods can be selectively employed.

To be specific, selection of a cDNA clone by an immunoscreeningtechnique using a specific antibody against the protein produced by thecDNA, the plaque hybridization or colony hybridization technique using aprobe having a selective binding affinity for the objective DNAsequence, or a combination thereof can be mentioned by way of example.

As to the probe to be used in the above procedure, it is generallyadvantageous to use a DNA chemically synthesized according to thenucleotide sequence information on the gene of the present invention butthe very gene of the invention which has already been acquired or afragment thereof can of course be used with advantage as said probe.Furthermore, the sense primer and antisense primer established based onthe nucleotide sequence information on the p51 gene of the presentinvention can be used as the screening probe.

For acquisition of the gene of the invention, DNA/RNA amplification bythe PCR method [Science, 230, 1350 (1985)] or a modification thereof canalso be used with advantage. Particularly under circumstances where afull-length cDNA can hardly be obtained from a library, the RACE [rapidamplification of cDNA ends] method [Jikken Igaku (ExperimentalMedicine), 12(6): 35 (1994)], in particular the 5′-RACE method [Frohman,M. A., et al., Proc. Natl. Acad. Sci., USA., 8: 8998 (1988)], can beused with advantage.

The primers for use in such PCR methods can be judiciously establishedaccording to the sequence information on the p51 gene of the inventionwhich has been uncovered in accordance with the present invention andcan be synthesized by the conventional procedure. Isolation andpurification of the amplified DNA or RNA fragment can be carried out bythe conventional techniques as mentioned hereinbefore, for example bygel electrophoresis or hybridization.

The nucleotide sequence of the p51 gene of the present invention or anyof the various DNA fragments which can be obtained as above can bedetermined in the routine manner, for example by the dideoxy method[Proc. Natl. Acad. Sci., USA., 74: 5463 (1977)], the Maxam-Gilbertmethod [Methods in Enzymology, 65: 499 (1980)] or, more expediently, bymeans of a commercial sequencing kit.

With the p51 gene of the present invention, for example by utilizing apartial or full-length nucleotide sequence of this gene, the expressionor non-expression of the p51 gene of the present invention in a human orother individual body or various tissues thereof can be specificallydetected.

This detection can be made in the routine manner. For example,determination at the cellular level by RNA amplification by RT-PCR[reverse transcribed-polymerase chain reaction; E. S. Kawasaki, et al.,Amplification of RNA In PCR Protocol, A Guide to methods andapplications, Academic Press, Inc., San Diego, 21-27 (1991)], Northernblotting analysis [Molecular Cloning, Cold Spring Harbor Lab. (1989)],in situ RT-PCR [Nucl. Acids Res., 21, 3159-3166 (1993)] or in situhybridization, the NASBA method [nucleic acid sequence-basedamplification, Nature, 350, 91-92 (1991)] and other techniques can bementioned. The preferred is the RT-PCR-SSCP method.

The primers for use in the PCR procedure are not particularly restrictedinasmuch as the p51 gene: (inclusive of a partial DNA) of the presentinvention can be specifically amplified, and can be judiciouslyestablished on the basis of the sequence information on the p51 gene ofthe invention. Usually, for example, primers each having a partialsequence of the p51 gene of the invention and a length ranging fromabout 10 to 35 nucleotides, preferably 15˜30 nucleotides, can beemployed.

Thus, the gene of the present invention includes DNA fragments which canbe used as specific primers and/or specific probes for detection of thehuman p51 gene of the invention.

Such a DNA fragment can be defined as the DNA characterized by itscapability to hybridize with a DNA having said nucleotide sequence(145-1488) under stringent conditions. The stringent conditionsmentioned above may be the conventional conditions used for primers andprobes and, for that matter, not particularly restricted but theabove-mentioned conditions, namely 0.1% SDS-containing 0.2×SSC at 50° C.or 0.1% SDS-containing 1×SSC at 60° C., can for example be mentioned.

With the human p51 gene of the present invention, the protein comprisingthe corresponding gene product (p51 protein) can be produced easily, ona large scale, and with good reproducibility by utilizing theconventional genetic engineering technology.

(2) The p51 Protein

The present invention, therefore, provides the p51 protein encoded bythe above-described gene of the invention.

As specific examples of the protein of the present invention, there canbe mentioned the p51A protein having the amino acid sequence shown underSEQ ID NO:1 and the protein designated as the p51B protein which has theamino acid sequence shown in SEQ ID NO:4. It should, however, beunderstood that the protein of the present invention is not limited tosaid p51A protein and p51B protein but includes their homologs. Thehomolog in this context includes the protein having an amino acidsequence derived from each of said amino acid sequences by deletion,substitution or addition of one˜a few or a plurality of amino acids andhaving said p51 activity. More particularly, the gene products of saidp51 gene homologs (p51-related genes including splicing variants andalleles) can be mentioned.

The protein of the present invention can be prepared by the conventionalrecombinant DNA technology [e.g. Science, 224, 1431 (1984); Biochem.Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA., 80,5990 (1983), etc.] based on the human p51 gene sequence informationprovided by the present invention.

(3) The Polypeptide Containing One or More Functional Domains of the p51Protein

The present invention is further directed to a polypeptide containing apartial region of said p51 protein.

The polypeptide preferably has the amino acid sequence corresponding toany of said various functional regions of the p51 protein and,specifically, there can be mentioned a polypeptide having an amino acidsequence corresponding to at least one domain selected from the groupconsisting of the transcriptional activation domain, DNA binding domainand oligomerization domain of the p51 protein.

As mentioned above, the locations of the transcriptional activationdomain, DNA binding domain and oligomerization domain of the p51 proteincan be identified by the amino acid numbers 1˜59, the amino acid numbers142˜321 and the amino acid numbers 359˜397, respectively, of the aminoacid sequence of the p51A protein which is shown under SEQ ID NO:1.

Therefore, the polypeptide of the present invention includes thefollowing.

-   (i) A polypeptide having the amino acid sequence corresponding to    the amino acid numbers 1˜59 of SEQ ID NO:1 (hereinafter referred to    briefly as amino acid sequence 1 (1-59)) and its equivalent.

The equivalent mentioned just above includes any polypeptide having anamino acid sequence derived from said amino acid sequence 1 (1-59) bydeletion, substitution or addition of one or a plurality of amino acidsand having a transcriptional activation function. The extent ofmodification or mutation of the amino acid sequence is not particularlyrestricted inasmuch as the modified polypeptide retains saidtranscriptional activation function. Preferably, however, thehomogeneity of the sequence so modified to the amino acid sequence 1(1-59) is not less than about 35%, particularly not less than 45%.

-   (ii) A polypeptide having an amino acid sequence identified by the    amino acid numbers 142˜321 of SEQ ID NO:1 (hereinafter referred to    briefly as amino acid sequence 1 (142-321)) and its equivalent.

The equivalent mentioned above includes any polypeptide having an aminoacid sequence derived from the amino acid sequence 1 (142-321) bydeletion, substitution or addition of one or a plurality of amino acidsand having said DNA binding function. The extent of modification ormutation of the amino acid sequence is not particularly restrictedinasmuch as the sequence as modified retains DNA binding activity butthe sequence preferably has a homology of not less than about 88%, morepreferably not less than 90% with respect to the amino acid sequence 1(142-321).

-   (iii) A polypeptide having an amino acid sequence identified by the    amino acid numbers 353˜397 of SEQ ID NO:1 (hereinafter referred to    briefly as amino acid sequence 1 (353-397) and its equivalent.

This equivalent includes a polypeptide having an amino acid sequencederived from said amino acid sequence 1 (353-397) by deletion,substitution or addition of one or a plurality of amino acids and havingan oligomerization function, for example the oligomerization domain in abroad sense (i.e. the amino acid numbers 353˜641 of SEQ ID NO:4) of thep51 protein. The extent of modification or mutation of the amino acidsequence is not particularly restricted inasmuch as the sequence somodified retains the oligomerization function but preferably retains ahomology of not less than about 70%, more preferably not less than 80%,with respect to the amino acid sequence 1 (353-397).

The polypeptide of the present invention may be a polypeptide whichcontains any of said amino acid sequence 1 (1-59) or an equivalentthereof, said amino acid sequence 1 (142-321) or an equivalent thereof,and said amino acid sequence 1 (353-397) or an equivalent thereof in oneregion or a polypeptide which contains two or more of said amino acidsequences in an optional combination either as a continuous region or adiscontinuous region.

The present invention further includes genes (DNA) havingoligonucleotide sequences coding for such polypeptides. Moreparticularly, the nucleotide sequence coding for said amino acidsequence 1 (1˜59) is the nucleotide sequence corresponding to thenucleotide numbers 145˜321 of SEQ ID NO:2; the nucleotide sequencecoding for said amino acid sequence 1 (142-321) is the nucleotidesequence corresponding to the nucleotide numbers 568˜1107 of SEQ IDNO:2; and the nucleotide sequence coding for said amino acid sequence 1(353-397) is the nucleotide sequence corresponding to the nucleotidenumbers 1201˜1335 of SEQ ID NO:2.

(4) Method of Producing the p51 Protein and the Materials for Use in itsProduction

The present invention further provides a method of producing said p51protein and the materials to be used for its production, for example, avector harboring said gene and host cells transformed with said vector.

More particularly, the production of said protein is carried out inaccordance with the procedure which comprises constructing a recombinantvector (expression vector) in which the gene coding for the desiredprotein may be expressed, transforming host cells with the resultingconstruct, culturing the transformant thus obtained, and harvesting thedesired protein from the culture obtained.

As said host cells, whichever of eucaryotic cells and procaryotic cellscan be employed.

The eucaryotic host cells include cells of vertebrae and yeasts, amongothers. Among the former cells, the monkey cell line COS [Cell, 23: 175(1981)], Chinese hamster ovarian cells and the dihydrofolatereductase-defective line thereof [Proc. Natl. Acad. Sci., USA., 77: 4216(1980)] can be mentioned as examples. As to the latter cells, cells ofyeasts belonging to the genus Saccharomyces can be mentioned as examplesbut these are not exclusive choices.

As the procaryotic host, various procaryotes which are commonlyemployed, such as Escherichia coli and Bacillus subtilis, can beliberally employed. The preferred host cells are those derived fromEscherichia coli, particularly cells of E. coli K12.

The expression vector is not particularly restricted inasmuch as itharbors the gene of the present invention and permits expression of saidgene but is generally selected with reference to the kinds of hostcells.

When cells of a vertebrate are used as host cells, generally anexpression vector having a promoter region upstream of the gene of theinvention, RNA splicing site, polyadenylation site and transcriptiontermination sequence can be used and, where necessary, it may furtherinclude a replication origin. As an example of such expression vector,pSV2dhfr [Mol. Cell. Biol., 1, 854 (1981)] having the early promoter ofSV40 can be mentioned.

When cells of eucaryotic microorganisms such as yeasts are used as hotcells, the expression vector which can be used includes pAM82 [Proc.Natl. Acad. Sci., USA., 80, 1 (1983)] which has the promoter of the acidphosphatase gene, and the vector for use in the present invention can beprepared by inserting the gene of the invention upstream of thispromoter. Preferably, a fusion vector obtainable by hybridization with aprocaryotic gene can be used and, as specific examples of such vector,pGEX-2TK and pGEX-4T-2 each having a GST domain with a molecular weightof 26000 (derived from S. japonicum) can be mentioned.

When procaryotic cells are used as host cells, the expression vector mayfor example be a vector equipped with a promoter region and SD(Shine-Dalgarno) sequence upstream of the gene so that the gene may beexpressed therein and further an initiation codon (e.g. ATG) necessaryfor the initiation of protein synthesis. Particularly when cells ofEscherichia coli (e.g. Escherichia coli K12) are used as host cells,generally pBR322 as such or modified is often used as the vector.However, these are not exclusive choices but other known bacterialstrains and known vectors can also be employed. As the promoter,tryptophan (trp) promoter, lpp promoter, lac promoter, PL/PR promoter,etc. can be employed.

The method of introducing said expression vector into the host cell(transformation method) is not particularly restricted, either, butvarious standardized methods can be utilized.

Culture of the resultant transformant can also be performed in theroutine manner. By such culture, the object protein encoded by the geneof the invention is expressed, produced, and accumulated in thetransformant cell or secreted extracellularly or on the cell membrane.

The medium for said culture can be judiciously selected from among theconventional culture media according to the type of host cells adopted,and culture can also be carried out under conditions suited for growthof the host cells.

The recombinant protein thus produced can be optionally isolated andpurified by various isolation procedures utilizing its physical,chemical or other properties [Seikagaku (Biochemical) Data Book II, pp.1175-1259, 1st Ed., 1st Impression, Jun. 23, 1980, Tokyo Kagaku Dojin;Biochemistry, 25(25): 8274 (1986); Eur. J. Biochem., 163: 313 (1987);etc.].

The procedures mentioned above specifically include the standardreconstitution treatment, treatment with a protein precipitating agent(salting out), centrifugation, osmotic shock method, sonic disruption,ultrafiltration, various kinds of chromatography, e.g. molecular sieveschromatography (gel filtration), adsorption chromatography, ion exchangechromatography, affinity chromatography, high performance liquidchromatography (HPLC), etc., dialysis, and their combinations. Theparticularly preferred procedure is affinity chromatography using acolumn conjugated with a specific antibody against the protein of theinvention.

In the designing of the object gene coding for the protein of theinvention, the nucleotide sequence of the human p51A gene as identifiedby the nucleotide sequence (145-1488) in SEQ ID NO:2 or the nucleotidesequence of the human p51B gene as identified by the nucleotide sequence(145-2067) in SEQ ID NO:5 can be utilized with advantage. If desired,this gene may be used with the codons designating respective amino acidresidues judiciously changed.

Furthermore, the partial modification of the amino acid sequence encodedby the human p51A gene or human p51B gene by the substitution, deletionor addition of some of the amino acid residues or a given partialsequence can be achieved by the various techniques mentionedhereinbefore, for example by site-specific mutagenesis.

The protein of the present invention can be synthesized by the standardtechnology for chemical synthesis in accordance with the amino acidsequence shown under SEQ ID NO:1 or the amino acid sequence shown underSEQ ID NO:4. This technology includes the liquid-phase and solid-phasemethods for peptide synthesis.

More particularly, the synthetic technology includes the so-calledstepwise elongation method in which one amino acid after another issequentially coupled together in accordance with the amino acid sequenceinformation and the fragment condensation method in which fragments eachconsisting of several amino acids are synthesized in advance and thencoupled together. The polypeptide of the present invention can besynthesized by whichever of the above alternative methods.

The condensation method for use in the above peptide synthesis may alsobe the conventional one, which includes but is not limited to the azidemethod, mixed acid anhydride method, DCC method, activated ester method,redox method, DPPA (diphenylphosphoryl azide) method, DCC+additive(1-hydroxybenzotriazole, N-hydroxysuccinamide,N-hydroxy-5-norbornene-2,3-dicarboximide or the like) method, andWoodward's method.

The solvent for use in these methods can also be judiciously selectedfrom among the common solvents which are well known to those skilled inthe art of peptide condensation. As examples, N,N-dimethylformamide(DMF), dimethyl sulfoxide (DMSO), hexaphosphoramide, dioxane,tetrahydrofuran (THF), ethyl acetate, etc. and mixed solvents thereof.

The carboxyl groups of amino acids or peptides which are not to beinvolved in the reaction for said peptide synthesis can be protectedgenerally by esterification, for example in the form of a lower alkylester, e.g. methyl ester, ethyl ester, tert-butyl ester or the like, oran aralkyl ester, e.g. benzyl ester, p-methoxybenzyl ester,p-nitrobenzyl ester or the like.

The amino acid having a functional group in its side chain, for examplethe hydroxyl group of the tyrosine residue, may be protected with anacetyl, benzyl, benzyloxycarbonyl, tert-butyl or other group, althoughthis protection is not indispensable. Furthermore, the guanidino groupof an arginine residue, for instance, can be protected with a suitableprotective group such as nitro, tosyl, p-methoxy-benzenesulfonyl,methylene-2-sulfonyl, benzyloxycarbonyl, isobornyloxycarbonyl,adamantyloxycarbonyl or the like.

The deprotection reactions of such protected amino acids, peptides andend product protein of the present invention for removal of theprotective groups can also be carried out by the conventional method,for example the catalytic reduction method or the method using liquidammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride,trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid orthe like.

The protein thus produced can be purified by the procedure which isconventionally utilized in the field of peptide chemistry, such as saidvarious methods, such as ion exchange chromatography, partitionchromatography, gel permeation chromatography, countercurrentdistribution, etc.

The protein of the present invention can bemused with advantage as animmunogen in the preparation of a specific antibody to the p51 protein,and by using such an immunogen, the desired antiserum (polyclonalantibody) and monoclonal antibody can be acquired.

The antibody production technology as such is well understood by thoseskilled in the art and, in the practice of the present invention, too,the conventional methods can be utilized [e.g. Zoku Seikagaku JikkenKoza (Supplemental Biochemical Experimental Series), Methods forImmunobiochemical Research, ed. by Japanese Biochemical Society (1986)].The antibody thus obtained can be used with advantage, for example inthe purification of the p51 protein and the immunological assay orcharacterization of the protein.

Furthermore, the protein of the present invention finds application inthe pharmaceutical field, in the manufacture of pharmaceutical productscontaining it as an active component.

(5) Pharmaceutical Compositions Containing the p51 Protein

The present invention, therefore, is further directed to saidpharmaceutical products containing the protein of the invention.

The protein mentioned above includes its pharmaceutically acceptablesalt. Such salt includes nontoxic alkali metal, alkaline earth metal andammonium salts, such as sodium, potassium, lithium, calcium, magnesium,barium and ammonium salts. Furthermore, said salt includes nontoxic acidaddition salts obtainable by reacting the peptide of the invention witha suitable organic or inorganic acid. The representative nontoxic acidaddition salts are the hydrochloride, hydrobromide, sulfate, bisulfate,acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate,phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate,succinate, tartrate, sulfonate, glycolate, ascorbate, benzenesulfonateand naphthalate, among others.

The present invention further comprises a pharmaceutical composition ordosage form which contains a pharmacologically effective amount of theprotein of the invention as an active ingredient together with asuitable nontoxic pharmaceutical carrier or diluent.

The pharmaceutical carrier which can be utilized in said pharmaceuticalcomposition (or dosage form) includes the diluents and excipients whichare commonly used according to the mode of use of the pharmaceuticalpreparation, such as filler, volume builder, binder, humectant,disintegrator, surfactant, lubricant, etc., and these are selectivelyemployed according to the unit dosage form of the pharmaceuticalpreparation.

The particularly preferred pharmaceutical preparation of the presentinvention is produced by using various formulating substances which canbe incorporated in the conventional protein preparation, such as thestabilizer, bactericide, buffer, isotonizing agent, chelating agent, pHcontrol agent, surfactant, etc., in suitable proportions.

The stabilizer mentioned above includes but is not limited to humanserum albumin, ordinary L-amino acids, saccharides and cellulosederivatives and these can be used independently or in combination with asurfactant or the like. Particularly in the combination use, thestability of the active ingredient can be further improved in certaincases.

The L-amino acids mentioned above are not particularly restricted butmay be glycine, cysteine, glutamic acid and so on.

The saccharides mentioned above include monosaccharides such as glucose,mannose, galactose, fructose, etc., sugar alcohols such as mannitol,inositol, xylytol, etc., disaccharides such as sucrose, maltose,lactose, etc., polysaccharides such as dextran, hydroxypropylstarch,chondroitin sulfate, hyaluronic acid, etc., and their derivatives.

The surfactant is not particularly restricted, either. Thus, ionicsurfactants and nonionic surfactants, such as surfactants in thepolyoxyethylene glycol sorbitan alkyl ester, polyoxyethylene alkylether, sorbitan monoacyl ester and fatty acid glyceride series can bementioned.

The cellulose derivatives are not particularly restricted, either, butmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose,carboxymethylcellulose sodium, etc. can be mentioned.

The suitable level of addition of the saccharide per 1 μg of the activeingredient is not less than about 0.0001 mg, preferably about 0.01˜10mg. The level of addition of the surfactant per 1 μg of the activeingredient may suitably be not less than about 0.00001 mg, preferablyabout 0.0001˜0.01 mg. The level of addition of human serum albumin per 1μg of the active ingredient may suitably be not less than about 0.0001mg, preferably somewhere within the range of about 0.001˜0.1 mg. Thelevel of addition of said amino acid per μg of the active ingredient issuitably about 0.001˜10 mg. The level of addition of the cellulosederivative per μg of active ingredient is suitably not less than about0.00001 mg, preferably about 0.001˜0.1 mg.

The amount of the active ingredient in the pharmaceutical dosage formcan be liberally selected from a broad range but can be judiciouslyselected from the range of generally about 0.00001˜70 weight %,preferably about 0.0001˜5 weight %.

The pharmaceutical dosage form of the present invention may besupplemented with various additives, such as a buffer, an isotonizingagent, a chelating agent, etc. The buffer mentioned above includes boricacid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid,glutamic acid, etc. and/or the corresponding salts (alkali metal oralkaline earth metal salts, e.g. sodium salt, potassium salt, calciumsalt, magnesium salt, etc.). The isotonizing agent includes but is notlimited to sodium chloride, potassium chloride, sugars and glycerin. Thechelating agent includes sodium edetate and citric acid, among others.

The pharmaceutical composition of the present invention can be used notonly as it is in the form of a solution but also provided in the form ofa lyophilized product which can be preserved and extemporaneouslyreconstituted with water or a buffer solution inclusive of physiologicalsaline to a suitable concentration.

The unit dosage form for the pharmaceutical composition of the presentinvention can be selected from various alternatives according to thetherapeutic purpose, and includes solid dosage forms, such as tablets,pills, powders, fine powders, granules, capsules, etc. and liquid dosageforms, such as solutions, suspensions, emulsions, syrups and elixirs.These dosage forms can be further classified as the oral, parenteral,transnasal, vaginal, rectal (suppository) and sublingual dosage forms,ointments and other products, and each product can be manufactured inaccordance with the established formulation and molding/processingprocedure.

Taking the manufacture of tablets as an example, the pharmaceuticalcarrier which can be used includes various excipients such as lactose,sucrose, sodium chloride, glucose, urea, starch, calcium carbonate,kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc.;binders such as water, ethanol, propanol, simple syrup, glucosesolution, starch solution, gelatin solution, carboxymethylcellulose,hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, etc.disintegrators such as carboxymethylcellulose sodium,carboxymethylcellulose calcium, low-substitution-degreehydroxypropylcellulose, dry starch, sodium alginate, agar powder,laminaran powder, sodium hydrogencarbonate, calcium carbonate, etc.;surfactants such as polyoxyethylene sorbitan fatty acid esters, sodiumlauryl sulfate, stearyl monoglyceride, etc.; disintegration inhibitorssuch as sucrose, stearin, cacao butter, hydrogenated oil, etc.;absorption promoters such as quaternary ammonium bases, sodium laurylsulfate, etc.; humectants such as glycerin, starch, etc.; adsorbentssuch as starch, lactose, kaolin, bentonite, colloidal silica, etc.; andlubricants such as purified talc, stearate salts, boric acid powder,polyethylene glycol and so on.

Furthermore, tablets may optionally be coated with a usual coatingmaterial to provide sugar-coated tablets, gelatin-coated tablets,enteric-coated tablets, film-coated tablets, etc. or even processed intomultilayer tablets such as double-layer tablets.

Pills can be manufactured by using various pharmaceutical carriersinclusive of excipients such as glucose, lactose, starch, cacao butter,hydrogenated vegetable oil, kaolin, talc, etc.; binders such as gumArabic, tragacanth powder, gelatin, ethanol, etc.; and disintegratorssuch as laminaran, agar and so on.

Capsules can be prepared by blending the active ingredient of thepresent invention with said various pharmaceutical carriers and fillingcapsule shells, such as hard gelatin capsule shells or soft capsuleshells, with the resulting composition.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable solutions, emulsions, syrups, elixirs, etc. as prepared byusing the conventional inert diluent, such as water, and may furthercontain auxiliary agents such as wetting agents, emulsifiers andsuspending agents. These dosage forms can be manufactured by theconventional procedure.

Liquid dosage forms for parenteral or nonoral administration, such assterile aqueous or nonaqueous solutions, emulsions and suspensions, canbe manufactured using a diluent such as water, ethyl alcohol, propyleneglycol, polyethylene glycol, ethoxylated isostearyl alcohol,polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acidester, a vegetable oil, e.g. olive oil, and may be formulated with aninjectable organic ester, such as ethyl oleate. Furthermore, suchpreparations may be supplemented with the conventional solubilizer,buffer, wetting agent, emulsifier, suspending agent, preservative,dispersant and other additives.

Sterilization may be carried out by filtration through a bacterialfilter, formulation of a bactericide, irradiation, heating or the like.Furthermore, said preparations can be processed into sterile soliddosage forms which can be extemporaneously dissolved in sterile water ora suitable sterilizable medium.

In the manufacture of rectal suppositories or a dosage form for vaginaladministration, there can be employed a pharmaceutical ointment basesuch as polyethylene glycol, cacao butter, a higher alcohol, a higheralcohol ester, gelatin, semisynthetic glyceride or the like.

In the manufacture of ointments inclusive of pastes, creams and gels,there can be employed such diluents as white petrolatum, paraffin,glycerin, cellulose derivatives, propylene glycol, polyethylene glycol,silicone oil, bentonite and vegetable oils such as olive oil.

Compositions for transnasal or sublingual administration can be preparedwith the well-known standard excipient in the conventional manner.

Where necessary, coloring agents, preservatives, flavoring agents,corrigents, sweeteners, and other medicinal substances can beincorporated in the pharmaceutical products of the present invention.

The method of administering said pharmaceutical product is notparticularly restricted but should be judiciously selected according tothe dosage form, patient factors such as age and sex, severity ofillness and other factors. For example, said tablets, pills, solutions,suspensions, emulsions, granules and capsules are administered orally.The parenteral product is used alone or mixed with a conventionalinfusion, such as a glucose or amino acid infusion, and administeredintravenously or, where necessary, administered alone intramuscularly,intradermally, subcutaneously or intraperitoneally. The suppositoriesare administered rectally; the vaginal preparation is administered intothe vagina. The transnasal preparation is administered into thenostrils; sublingual tablets are administered buccally; and ointmentsare topically administered transdermally.

The amount of the protein of the invention in the pharmaceutical productand the dosage thereof are not particularly restricted but canbe-judiciously selected from a broad range according to the expectedtherapeutic effect, administration method, treatment course or duration,patient factors such as age and sex, and other factors. Generally, thedosage is usually about 0.01 μg˜10 mg/kg body weight/day, preferablyabout 0.1 μg˜1 mg/kg b.d./day, and this amount can be administered onceor in a few divided doses daily.

(6) Gene Therapy

The present invention further provides a method of gene therapyutilizing the human p51 gene of the invention. This therapeutic methodmay be regarded as a method for imparting the wild type p51 genefunctions to the cells harboring a mutant p51 gene. By imparting thenormal functions inherently possessed by the wild type p51 gene or geneproduct to cells, neoplastic growth of the recipient/target cells can beinhibited. The wild type p51 gene mentioned above can be transferredinto the objective cells by means of a vector or plasmid capable ofmaintaining the gene extrachromosomally. In this case, the particulargene is expressed from outside of the chromosome.

In introducing the wild type p51 gene into cells harboring such a mutantp51 gene to have a normal p51 protein expressed in the recipient cells,the p51 gene need not have a full-length sequence but may be saidmodified gene insofar as the latter has substantially homologous desiredfunctions with respect to the unmodified gene. As an alternative, a genehaving a partial sequence retaining certain such functions can beemployed. As an example of the gene mentioned just above, there can bementioned a gene coding for a portion of p51 protein which is necessaryfor nontumorous growth of cells (cell growth inhibition).

The wild type p51 gene or a fragment thereof is preferably introducedinto mutant cells in such as manner that a recombination will take placewith the endogenous mutant p51 gene. For such a recombination,occurrence of a double recombination correcting for the p51 genemutation is said to be required.

The vectors which can be used in the transfer of the object gene forboth such recombination and extrachromosomal maintenance of the gene arealready known in the art and any of the known vectors can be used in thepractice of the present invention. For example, a virus vector orplasmid vector which harbors a copy of p51 gene linked to an expressioncontrol element and is capable of insuring expression of the geneproduct within the target-cells can be mentioned. As such a vector, theexpression vectors mentioned above can be generally used but preferablyvectors constructed using such source vectors as the vectors disclosedin U.S. Pat. No. 5,252,479 and PCT WO 93/07282 (pWP-7A, pwP-19, pWU-1,pWP-8A, pWP-21 and/or pRSVL, among others) or pRC/CMV (Invitrogen). Themore preferred are the various virus vectors described hereinafter.

As the promoter for the vector to be used in gene therapy, the promotersintrinsic to the target affected tissues in various diseases can beutilized with advantage.

Specific examples may be cited below. For the liver, for instance,albumin, α-fetoprotein, α1-antitrypsin, transferrin, transthyretin, etc.can be mentioned by way of example. For the colon, carboxyl anhydrase I,carcinoembrogen antigen, etc. can be mentioned. For the uterus andplacenta, estrogen, aromatase cytochrome P450, cholesterol side-chaincleaving P450, 17α-hydroxylase P450, etc. can be mentioned.

For the prostate, prostatic antiegn, gp91-fox gene, prostate-specifickallikrein, etc. can be mentioned. For the mamma, erb-B2, erb-B3,β-casein, β-lactoglobin, whey protein, etc. can be mentioned. For thelung, the activator protein C uroglobulin, among others, can bementioned. For the skin, K-14-keratin, human keratin 1 or 6, leucrin,etc. can be mentioned.

For the brain, neuroglia fiber acid protein, mature astrocyte-specificprotein, myelin, tyrosine hydroxylase pancreatic villin, glucagon,Langerhans islet amyloid polypeptide, etc. can be mentioned. For thethyroid, thyroglobin, calcitonin, etc. can be mentioned. For the bone,α1 collagen, osteocalcin, bone sialoglycoprotein, etc. can be mentioned.For the kidney, renin, liver/bone/kidney alkaline phosphatase,erythropoietin, etc. can be mentioned. For the pancrease, amylase, PAP1,etc. can be mentioned.

The gene (the whole or a fragment) to be used for the construction of agene transfer vector can be easily produced and acquired by the standardgenetic engineering technology based on the nucleotide sequenceinformation about the p51 gene of the invention as mentionedhereinbefore.

The introduction of such a gene transfer vector into cells can becarried out by various alternative techniques known to those skilled inthe art, such as electroporation, calcium phosphate coprecipitation,viral transduction and so on. The cells transformed with the wild typep51 gene can be used as they are in the isolated form as the agent fortumor suppression or inhibition of cancer metastasis or as a modelsystem for therapeutics research.

In gene therapy, said gene transfer vector can be introduced into thetumor cells of a patient by topical administration to the tumor site orby systemic administration to the patient by injection. By systemicadministration, the gene can be caused to arrive at any tumor cellsmetastable to other sites. If the transformed cells cannot bepermanently taken up in the chromosomes of the target tumor cells, theabove administration may be repeated periodically.

The method of gene therapy according to the present invention includesboth the in vivo method which comprises administering a material forsaid gene transfer (gene transfer vector) directly into the body and theex vivo method which comprises withdrawing the target cells from thepatient's body, introducing the gene extracorporeally, and returning thecells into the body.

A further possible alternative is a gene therapy using a ribozyme whichcomprises introducing the human p51 gene directly into cells andcleaving the RNA chain with the ribozyme which is an active molecule.

The gene transfer vector harboring the human p51 gene of the inventionor a fragment thereof and a gene-therapeutic agent comprising cellstransformed with the human p51 gene by means of said vector as an activeingredient are directed especially to the therapy of cancers but thegene therapy (treatment) mentioned above can be applied also to thetherapy of hereditary diseases and viral diseases such as AIDS, as wellas for the purpose of gene labeling.

The target cells to which the gene is transferred can be judiciouslyselected according to the object of gene therapy (treatment). Forexample, as the target cells, not only cancer cells and tumor tissuesbut also lymphocytes, fibroblasts, hepatocytes, hematopoietic stem cellsand other cells can be mentioned.

The method of introducing the gene into cells in the gene therapyincludes a viral transfer method and a non-viral transfer method.

As the viral transfer method, the method using a retrovirus vector, forinstance, can be used in view of the fact that the human p51 gene is aforeign gene which is expressed in normal cells. As other virus vectors,adenovirus vector, HIV (human immunodeficiency virus) vector,adeno-associated virus (AAV) vector, herpes virus vector, herpes simplexvirus (HSV) vector and Epstein-Barr virus (EBV) vector, etc. can bementioned.

The non-viral gene transfer method includes the calcium phosphatecoprecipitation method; the membrane-fusion liposome method whichcomprises fusing DNA-containing liposomes with an inactivated Sendaivirus as exposed to UV radiation for gene destruction to constructmembrane-fusion liposomes and introducing the DNA into cells by directfusion with the cell membrane [Kato, K., et al., J. Biol. Chem., 266,22071-22074 (1991)]; the method which comprises coating the plasmid DNAwith gold and introducing the DNA physically into cells by high-voltagedischarge [Yang, N. S. et al., Proc. Natl. Acad. Sci., 87, 9568-9572(1990)]; the naked DNA method in which the plasmid DNA is directlyinjected into an organ or tumor in vivo [Wolff, J. A., et al., Science,247, 1465-1467 (1990)]; the cationic liposome method in which the geneentrapped in multilamellar positively-charged liposomes are introducedinto cells [Yagi, K., Advance in Medicine, vol. 175, No. 9, 635-637(1995)]; and the ligand-DNA complex method in which a ligand which bindsa receptor expressed on the target cells is coupled to the DNA so thatthe gene may be introduced exclusively into the selected cells and notinto other cells and the resulting complex is administered [Frindeis, etal., Trends Biotechnol., 11, 202 (1993); Miller, et al., FASEB J., 9,190 (1995)] among others.

The ligand-DNA complex method mentioned above includes the method whichcomprises using the asialoglycoprotein receptor expressed in liver cellsas the target and an asialoglycoprotein as the ligand [Wu, et al., J.Biol. Chem., 266, 14338 (1991); Ferkol, et al., FASEB J., 7, 1081-1091(1993)] and the method which comprises using the transferrin receptorexpressed at a high level in tumor cells as the target and transferrinas the ligand [Wagner et al., Proc. Natl. Acad. Sci., USA., 87, 3410(1990)], among others.

Furthermore, the gene transfer method which can be used may be asuitable combination of said-biological and physical gene transfermethods. As such a combination method, there can be mentioned the methodin which a plasmid DNA having a given size is used in combination with apolylysine-conjugated antibody specific to adenovirus hexon protein.According to this method, the complex formed is bound to the adenovirusvector and the resulting trimolecular complex can be used to infectcells and thereby transfer the gene of the present invention. Inaccordance with this method, effective binding, endogenization andendosome degradation can take place before the DNA coupled to theadenovirus vector is damaged. Moreover, said liposome/DNA complex maymediate the gene transfer in vivo.

The method of constructing a virus vector for transfer of the gene ofthe invention and the method of introducing the gene into the targetcells or target tissue are now described.

The retrovirus vector system comprises the virus vector and helper cells(packaging cells). The helper cells mentioned above are cells in whichgenes such as retrovirus structural protein gag (the structural proteinin the virus particle), pol (reverse transcriptase) and env (shellprotein) have been expressed but have not formed virus particles. On theother hand, the virus vector has a packaging signal and LTR (longterminal repeats) but are devoid of structural genes necessary for virusreplication, such as gag, pol and env. The packaging signal is asequence serving as the tag in the assembling of a virus particle andthe selective genes (neo, hyg) and the desired gene (p51 gene or afragment thereof) incorporated in the cloning site are inserted in placeof the virus gene. Here, in order to acquire a high titer of virusparticles, it is important to make the insert as short as possible,broaden the packaging signal by including a part of the gag gene and usecare not to leave the ATG of the gag gene.

By introducing the vector DNA harboring the object p51 gene into thehelper cells, the vector genome RNA is packaged with the virusstructural protein of the helper cells to form and secrete virusparticles. After the virus particle as the recombinant virus hasinfected the target cell, the DNA reverse-transcribed from the virusgenome RNA is integrated into the cell nucleus and the gene insertedinto the vector is expressed.

As the method of improving the efficiency of transfer of the objectgene, the method using a fragment containing the cell adhesion domain offibronectin, the heparin-binding site and conjugation segment can beemployed [Hanenberg, H., et al., Exp. hemat., 23, 747 (1995)].

As an example of the vector for use in the above retrovirus vectorsystem, the retrovirus derived from mouse leukemia virus [McLachlin, J.R., et al., Proc. Natl. Acad. Res. Molec. Biol., 38, 91-135 (1990)] canbe mentioned.

The method comprising using the adenovirus vector is now described indetail.

Construction of said adenovirus vector can be carried out in accordancewith the methods of Berkner [Berkner, K. L., Curr. Topics Microbiol.Immunol., 158, 39-66 (1992)], Setoguchi, Y. et al. [Setoguchi, Y., etal., Blood, 84, 2946-2953 (1994)], Kanegae, H. et al. [ExperimentalMedicine(?), 12, 28-34 (199.4)], and Ketner et al. [Ketner, G., et al.,Proc. Natl. Acad. Sci., USA., 91, 6186-6190 (1994)].

For example, for the construction of a nonproliferative adenovirusvector, the early gene E1 and/or E3 gene regions of adenovirus are firstremoved. Then, a plasmid vector harboring the object foreign geneexpression unit (consisting of the gene to be transferred, which is thep51 gene in the present invention, a promoter for transcription of thegene, and poly-A which imparts stability to the transcript) and aportion of the adenovirus genome DNA and a plasmid harboring theadenovirus genome are used to concurrently transfect cells, e.g.293-cells. By causing a homologous recombination to take place betweenthe two and thereby substitute the gene expression unit for E1, thenonproliferative adenovirus vector harboring the p51 gene according tothe present invention can be constructed. It is also possible tointegrate the adenovirus genome DNA into the cosmid vector to constructa 3′-end adenovirus vector with the terminal protein added. Furthermore,the YAC vector can also be utilized in the construction of a recombinantadenovirus vector.

The production of an adeno-associated virus (AAV) vector is now brieflydescribed. AAV was discovered as a small-sized virus contaminating aculture system of adenovirus. Of this virus, the parvovirus genus whichdoes not require a helper virus for replication but proliferatesautonomously in the host cell and the dependvirus which requires ahelper virus have been confirmed. This AAV is one of the common viruseswhich has a broad host range and infects a variety of cells. Its genomeis a linear single-stranded DNA consisting of 4680 nucleotides and the145 nucleotides at either terminus has a characteristic sequence calledITR (inverted terminal repeat). This ITR region is a replicationinitiation point and plays the role of a primer. Furthermore, this ITRis indispensable to the packaging to the virus particle and theintegration into the chromosomal DNA of the host cell. In addition, withregard to the virus protein, the left-half of the genome codes for anonstructural protein, that is the regulatory protein Rep which controlsreplication and transcription.

Construction of a recombinant AAV can be carried out by utilizing theproperty of AAV to be integrated with chromosomal DNA, whereby a vectorfor transfer of the desired gene can be constructed. More particularly,in accordance with this method, a plasmid (AAV vector plasmid) harboringthe object gene to be transferred (human p51 gene) inserted between theremnant ITRs at both the 5′- and 3′-ends of the wild type AAV is firstconstructed. On the other hand, the virus protein necessary for virusreplication and construction of the virus particle is supplied from anindependent helper plasmid. It is insured that there will be nonucleotide sequence common to both plasmids so that a recombinantwild-type virus will not emerge. Then, both plasmids are introduced bytransfection into, for example, 293-cells, which are further infectedwith adenovirus (which may be nonproliferative type when 293-cells areused) as the helper virus, whereby the objective recombinant AAV of thenonproliferative type is produced. Since this recombinant AAV exists inthe nucleus, it is recovered by freeze-thaw and the contaminantadenovirus is inactivated by heating at 56° C. Where necessary, therecombinant AAV is isolated and concentrated by ultracentrifugation withcesium chloride. In this manner, the objective recombinant AAV fortransfer of the object gene can be acquired.

Construction of the HIV vector can be carried out typically inaccordance with the method of Shimada et al. [Shimada, T., et al., J.Clin. Invest., 88, 1043-1047 (1991)].

Since the HIV virus specifically infects helper T cells with CD4 as thereceptor, a tissue-specific gene transfer HIV vector adapted forspecific introduction of a gene into human CD4-positive cells can beconstructed. This HIV vector is optimal for the gene therapy of AIDS.

Construction of a recombinant HIV vector can be carried out typically asfollows. First, the packaging plasmid CGPE is constructed in such amanner that the structural genes gag, pol and env and the control genes(tat, rev, etc.) necessary for expression thereof may be expressed withthe cytomegalovirus (CMV) promoter and the human globin gene poly Asignal (poly A). Then, the vector plasmid HXN can be constructed so asto permit efficient proliferation in COS cells by inserting thebacterial neomycin-resistant gene (neoR) having a promoter for thymidinekinase (TK) as a marker gene between the two LTRs of HIV and furtherinserting a SV40 replication mechanism into the basal plasmid vector. Asthe above packaging plasmid CGPE and vector plasmid HXN are concurrentlyintroduced by transfection into COS cells, the objective neoRgene-integrated recombinant virus is produced and released into theculture medium in a large quantity.

Production of the EBV vector can be carried out typically in accordancewith the method of Shimidzu et al [Shimidzu, N., Cell Engineering(?),14(3), 280-287 (1995)].

The production of an EBV vector for transfer of the gene of the presentinvention is now briefly described. EB virus (Epstein-Barr virus: EBV)is a virus belonging to the herpes family and was isolated from culturedcells derived from Burkitt lymphoma by Epstein and coworkers in 1964[Kieff, E. and Liebowitz, D.: Virology, 2nd ed. Raven Press, New York,1990, pp.1889-1920]. The EBV has cell-transforming activity and,therefore, in order that it may be utilized as a gene transfer vector,the virus defective of this tranforming activity must be prepared. Thiscan be done as follows.

Thus, in the first place, an EBV genome close to the target DNA withwhich the desired foreign gene is to be integrated is cloned. With thisclone, a foreign gene DNA fragment and a drug-resistant gene areintegrated to prepare a vector for production of a recombinant virus.Then, the vector for construction of a recombinant virus is excised withsuitable restriction enzymes and introduced by transfection intoEBV-positive Akata cells. The recombinant virus produced by thehomologous recombination can be recovered, together with the wild typeAkata EBV, through virus production stimulation by anti-surfaceimmunoglobulin treatment. This is used to infect EBV-negative Akatacells and a resistant strain is selected in the presence of the drug toobtain the desired Akata cells infected exclusively by the recombinantvirus and free from the wild type EBV. Then, by inducing virus activityin the recombinant virus-infected Akata cells, the objective recombinantvirus vector can be produced in a large quantity.

Production of a non-virus vector for the introduction of the desiredgene into target cells without use of a recombinant virus vector can becarried out by the gene transfer technique using a membrane fusionliposome preparation. This is a technique such that by imparting fusionactivity to a membrane liposome (a vesicle having a lipid bilayerstructure), the contents of the liposome are directly introduced intothe cell.

Introduction of the gene by means of such membrane fusion liposomes canbe carried out typically in accordance with the method of Nakanishi etal. [Nakanishi, M., et al., Exp. Cell Res., 159, 399-499 (1985);Nakanishi, M., et al., Gene introduction into animal tissues. In Trendsand Future Perspectives in Peptide and Protein Drug Delivery (ed. byLee, V. H. et al.)., Harwood Academic Publishers Gmbh. Amsterdam, 1995,pp. 337-349].

This method of gene transfer by means of said membrane fusion liposomesis briefly described below. Thus, liposomes in which Sendai virus withits gene inactivated by UV irradiation, the object gene and a highmolecular substance, such as protein, have been entrapped is fused at37° C. This membrane fusion liposome has a structure called“pseudovirus” which consists of a liposome-derived inner cavity and anouter spike structure similar to that of the virus envelope. Afterpurification by sucrose density gradient centrifugation, the membranefusion liposomes are caused to get adsorbed on the target cultured cellsor tissue cells at 4° C. Then, as the temperature is increased to 37°C., the contents of the liposomes are introduced into the cell, wherebythe desired gene can be transferred to the target cells. The lipid forsaid liposome in this case is a synthetic phospholipid composed of 50%(by mole) each of cholesterol and lecithin and having a negative chargeand is preferably formed as a unilamellar liposome with a diameter of300 nm.

As an alternative method of introducing the gene into the target cell bymeans of liposomes, the gene transfer method using cationic liposomescan be mentioned. This method can be practiced in accordance with themethod of Yagi et al. [Yagi, K., et al., B. B. R. C., 196, 1042-1048(1993)]. Thus, with attention paid to the fact that plasmids and cellsare both negatively charged, a positive charge is imparted to both theexternal and internal surfaces of the liposome membrane so that theuptake of the plasmid is increased by static electricity to enhance theinteraction with the cells. The liposome used in this case is preferablya multilamellar large vesicle (MLV) having a positive charge, althoughit is possible to use a large unilamellar vesicle (LUV) or a smallunilamellar vesicle (SUV) to construct a complex with the plasmid forintroduction of the desired gene.

The method of preparing a plasmid-containing cationic MLV is brieflydescribed below.

In the first place, a chloroform solution containing the lipid TMAG(N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride), DLPC(dilauroyl phosphatidylcholine) and DOPE (dioleoylphosphatidylethanolamine) in a molar ratio of 1:2:2 is prepared (lipidconcentration: 1 mM). Then, a total of 1 μmol of lipid is placed in acentrifuge tube and the chloroform is distilled off under pressure usinga rotary evaporator to prepare a lipid thin film. The residualchloroform is completely removed under reduced pressure and the film isdried. Then, 0.5 ml of Mg and Ca-containing Dulbecco'sphosphate-buffered saline containing 20 μg of the gene transfer plasmidis added and, after nitrogen purging, the mixture is stirred with avortex mixer for 2 minutes to give a suspension of the gene-harboringplasmid-containing cationic MLV.

The following is an example of use of the plasmid-containing cationicMLV as a gene therapy agent. For example, the expression plasmidintegrated with the cDNA of the object gene is entrapped in an amount of0.6 μg as DNA per 30 nmole of liposome lipid in the above cationic MLVand the liposomes are suspended in 2 μl of phosphate-buffered saline.This suspension is administered to the target cells extracted from thepatient or the target patient tissue every other day.

In this connection, in the guidelines established by the Ministry ofHealth and Welfare of Japan, the gene therapy is defined as “toadminister a gene or a gene-integrated cell into the human body for thetherapy of a disease”. However, the gene therapy in the context of thepresent invention encompasses not only the therapy falling under theabove definition but also the therapy of various diseases inclusive ofcancer which comprises introducing a gene characterized as a tumorsuppressor gene, such as the human p51 gene, into said target cells andthe practice which comprises introducing a marker gene or cellsintegrated with such a marker gene into the human body.

In the gene therapy according to the present invention, the method ofintroducing the object gene into the target cells or tissues includesthe following two representative methods.

The first method comprises isolating the target cells from the patientto be treated, growing the cells extracorporeally, for example in thepresence of interleukin-2 (IL-2) or the like, introducing the p51 geneligated to the retrovirus vector into the cells, and retransplanting theresulting cells (ex vivo method). This method is suited for the therapyof ADA deficiency syndrome, hereditary diseases and cancers associatedwith defective genes, AIDS, and other diseases.

The second method is a direct gene transfer method which comprisesinjecting the object gene (human p51 gene) directly into the patient'sbody or target site, such as a tumor tissue (direct method).

More particularly, the above first method of gene therapy can be carriedout typically as follows. Thus, the mononuclear cells isolated from thepatient are separated from monocytes with a blood separator, theharvested cells are cultured in the presence of IL-2 in a suitablemedium such as AIM-V medium for about 72 hours, and the vector harboringthe gene to be introduced (human p51 gene) is added. For enhanced genetransfer efficiency, the system may be centrifuged at 2500 rpm in thepresence of protamine at 32° C. for 1 hour and incubated under 10%carbon dioxide gas at 37° C. for 24 hours. After the above procedure isrepeated a few times, the cells are further cultured in the presence ofIL-2 in AIM-V or other medium for 48 hours. The cells are washed withsaline, viable cells are counted, and the gene transfer efficiency isevaluated by carrying out said in situ PCR or, when the object functionis enzymatic activity, assaying the activity to confirm the genetransfer effect.

In addition, a safety check comprising culture of the bacteria and fungicontaminating the cultured cells and testing for mycoplasma infectionand for endotoxin is carried out to confirm safety. Then, the culturedcells integrated with a predicted effective dose of the gene (human p51gene) are returned to the patient by intravenous drip injection. Thisprocedure is repeated at an interval of a few weeks or a few months forgene therapy.

The dosage of the virus vector can be judiciously selected according tothe kind of target cell. Usually, in terms of virus titer, a dose withinthe range of 1×10³ cfu˜1×10⁸ cfu is used per 1×10⁸ target cells.

An alternative version of the above first method, which can be employed,comprises co-culturing virus-producing cells containing a retrovirusvector harboring the desired gene (human p51 gene) with, for example,the patient's cells to introduce the gene (human p51 gene) into thetarget cells.

In carrying out the second method of gene therapy (direct method), apreliminary experiment, particularly an ex vivo experiment, ispreferably performed to confirm whether the object gene (human p51 gene)may be actually transferred or not by a PCR of the vector gene cDNA oran in situ PCR assay or confirm the desired effect of therapy resultingfrom the transfer of the object gene (human p51 gene), for example anelevation in specific activity or an enhancement or suppression ofgrowth of target cells. Furthermore, when a virus vector is used, it is,of course, important, in conducting a gene therapy, to confirm thesafety of introduction of the gene by carrying out the PCR to search forproliferative retrovirus, determining the reverse transcriptaseactivity, or monitoring the membrane protein (env) gene by a PCRtechnique.

When the method of gene therapy according to the present invention isapplied to cancers or malignant tumors in particular, a typical protocolfor cancer therapy may comprise isolating cancer cells from the patient,treating the cells with an enzyme or the like to establish a culturedcell line, introducing the desired gene into the target cancer cells bymeans of retrovirus or the like, carrying out a screening with G418cells, determining the amount of expression of IL-12 or the like (invivo), subjecting the cells to radiation treatment, and inoculating thecells into the patient's tumor or paratumor.

The herpes simplex virus-thymidine kinase (HSV-TK) gene reportedlycauses cell death due to division aging, particularly by converting thenucleotide analog gancyclovir (GCV) to a toxic intermediate, and thereis known a gene therapy using this gene in tumors [U.S. Pat. No.5,631,236; J P Kohyo H9-504784]. This method is a method of gene therapywhich utilizes the phenomenon that when cells capable of producing aretrovirus vector harboring said HSV-TK gene, known as a suicide gene,are injected and, one week later, the antiviral agent GCV isadministered, the GCV in the gene-transformed cells is activated byphosphorylation to induce death of these cells and death of thesurrounding non-gene-transferred cells due to cell contact through thegap junction. The gene transfer vector of the invention or cellscontaining this vector can be used in the above gene therapy as well.

An alternative method of gene therapy comprises preparingimmunoliposomes containing the gene (human p51 gene) coupled to theantibody capable of coupling to the target cell surface to introduce theentrapped cDNA into the target cells selectively and with goodefficiency. Feasible as well is a gene therapy which comprisesadministering said cytokine gene-harboring virus vector and said suicidegene-harboring adenovirus at one and the same time. These methodsinvariably fall within the expertise of those skilled in the art.

(7) Pharmaceutical Composition for Gene Therapy

The present invention further provides a pharmaceutical composition oragent (gene-therapeutic agent) comprising a pharamcologically effectiveamount of cells to which either the gene transfer vector or the objectgene (e.g. human p51 gene or the like) of the invention has beentransferred as an active ingredient together with a suitable nontoxicpharmaceutical carrier or diluent.

The pharmaceutical carrier which can be formulated in the pharmaceuticalcomposition (preparation) of the present invention includes theconventional diluents and excipients, such as filler, volume builder,binder, humectant, disintegrator, surfactant, lubricant, etc., which arecommonly employed according to the method of use of the preparation andthese carriers can be selectively used with reference to the desiredunit dosage form.

The unit dosage form for the pharmaceutical composition of the presentinvention includes the same dosage forms as those mentioned for the p51protein and can be judiciously selected according to the therapeuticobjective.

For example, a pharmaceutical preparation containing the gene transfervector of the invention can be provided in the form of said vectorentrapped in liposomes or in the form of cultured cells infected with avirus containing a retrovirus vector harboring the object gene.

These can be prepared as solutions in phosphate-buffered saline (pH7.4), Ringer's injection or an intracellular fluid compositioninjection, for instance, or in such a form that it may be administeredtogether with a substance capable of enhancing the efficiency of genetransfer, such as protamine.

The method of administering the above pharmaceutical preparation is notparticularly restricted but is selected according to the dosage form,the patient's age, sex and other factors, the severity of illness, andother conditions.

The amount of the active ingredient to be incorporated in saidpharmaceutical composition or preparation are not particularlyrestricted but can be liberally selected from a broad range according tothe desired therapeutic effect, administration method, duration oftreatment, the patient background inclusive of age and sex, and otherconditions.

Generally speaking, the daily dose of the gene-harboring retrovirusvector as a pharmaceutical preparation per kilogram body weight may forexample be about 1×10³ pfu to 1×10¹⁵ pfu in terms of retrovirus titer.

In the case of cells to which the object gene has been introduced, thedosage may be judiciously selected from the range of about 1×10⁴cells/body to about 1×10¹⁵ cells/body.

The pharmaceutical product can be administered once a day or in a fewdivided doses a day, and may be administered intermittently, for exampleone to several weeks apart. Moreover, it can be advantageouslyadministered in combination with a substance capable of enhancing theefficiency of gene transfer, such as protamine, or a pharmaceuticalpreparation containing said substance.

When the gene therapy of the present invention is applied to thetreatment of a cancer, the various methods of gene therapy mentionedabove may be used in a suitable combination (combination gene therapy)and/or in combination with the conventional cancer chemotherapy,radiation therapy, immunotherapy and/or other therapy. Furthermore, thegene therapy according to the present invention can be carried out withreference to the NIH Guidelines, inclusive of the safety aspect thereof[cf. Recombinant DNA Advisory Committee, Human. Gene Therapy, 4, 365-389(1993)].

(8) Application to Tumor Diagnosis

In accordance with the present invention, the presence of a mutant p51gene which promotes tumorigenesis in human cells can be detected by theprocedure which comprises preparing a blood, serum or other biologicalsample, optionally: extracting the nucleic acid, and analyzing it forthe presence or absence of a sensitive mutant p51 gene. Furthermore, inaccordance with the present invention, the existence of a neoplasticchange in cells or tissues, a marker of progression to a prodromal stateof malignancy or a prognostic marker can be detected by the procedurewhich comprises preparing a disorder-containing biological sample andanalyzing it for the presence or absence of a neoplastic mutant p51gene. By the above procedure, the presence of a neoplasm in cells ortissues, a marker of progression to a prodromal state of malignancy or aprognostic marker can be detected, thus allowing to establish adiagnosis, for example the diagnosis of a cancer, evaluate the effect ofa cancer therapy, or predict the prognosis of the cases.

According to this detection method, based on the mutant p51 geneinformation obtained from a tumor-bearing patient sample, for example onthe information about the mutation site of the p51 gene and the mutantsequence information, the relevant mutant DNA fragment is prepared anddesigned so that it may be used in the screening for the mutant geneand/or the amplification thereof. More particularly, the probe for usein plaque hybridization, colony hybridization, Southern blotting,Northern blotting, etc. or the probe for PCR amplification of the mutantDNA fragment can be constructed. For such purposes, a primer having thesame sequence as the mutation is first prepared and reacted, as ascreening probe, with a biological sample (nucleic acid sample), wherebythe presence of a gene having a mutated p51 gene sequence can beconfirmed. To facilitate detection of the target sequence, said nucleicacid sample may be prepared by utilizing various techniques such aslysis, restriction enzyme digestion, electrophoresis or dot blotting.

Referring to the screening method mentioned above, the use of a PCRmethod is particularly preferred from the standpoint of sensitivity, andthis method is not particularly restricted inasmuch as the mutant p51fragments are used as primers. Thus, any of the hitehrto-knowntechniques [Science, 230, 1350-1354 (1985)] and versions of PCR whichhave been newly developed or will be used in future [Sakaki, Y et al.(ed): Experimental Medicine(?), Supplemental Issue, 8(9) (1990),Yōdo-sha; Protein, Nucleic Acid and Enzyme, Special Supplemental Issue,35(17) (1990), Kyoritsu Shuppan] can be employed.

The DNA fragments for use as primers are chemically synthesizedoligoDNAs, and these oligoDNAs can be synthesized by using an automatedDNA synthesizing hardware, such as the DNA synthesizer Pharmacia LKBGene Assembler Plus (Pharmacia). The length of the primer so synthesized(sense primer or antisense primer) is preferably the equivalent of about10˜30 nucleotides. The probe for use in said screening is usuallylabeled but may be an unlabeled one, and the detection may be madeeither directly or indirectly by specific binding with a labeled ligand.The suitable label and the method of labeling the probe or the ligandare known to those skilled in the art, and the radioactive label,biotin, fluorescent group, chemiluminescent group, enzyme, antibody,etc. which can be incorporated by the known techniques such as nicktranslation, random priming or kinase treatment are also included in therelevant technology.

The PCR method for use in said detection includes RT-PCR, for instance,and various modifications of PCR which are used in the art can beapplied likewise.

It is also possible to detect the wild type p51 gene and/or mutant p51gene and quantitate the DNAs of these genes. This technology includesbut is not limited to the competitive assay such as MSSA [Kinoshita, M.et al., CCA, 228, 83-90 (1994)] and PCR-SSCP which is known to be amutation detecting technique utilizing the change in mobility associatedwith a change in the higher-order structure of a single-stranded DNA[Orita, M. et al., Genomics, 5, 874-879 (1989)].

The above analytical methods mentioned by way of example can be carriedout as follows. For example, one or a plurality of primers containingthe mutation of p51 (e.g. the mutated sequence based on site mutationinformation obtained from a cancer patient or the like) are firstprepared and hybridized with the DNA obtained from a biological sample.Then, the mobility and peak area measured by SSCP analysis of thestandard wild type p51 DNA fragment are compared with the mobility andpeak area in the test sample as the product of amplification using saidprimers to thereby detect the mutation in a specific region of p51 andsimultaneously quantitate the product of mutation.

The test sample containing the mutant p51 gene to be measured is notparticularly restricted inasmuch as it contains said mutant gene, thusincluding various biological materials such as blood, serum, urine andexcised tissues. The mutant p51 gene can be extracted from such testsamples, purified and prepared in the routine manner. Therefore, bycomparing the mobility of said standard DNA fragment of the invention,as determined in advance, with the mobility of the amplification productin the test sample as obtained in the PCR amplification of the p51 DNAof the test sample using a mutant p51 primer pair, the mutation in aspecific region of p51 DNA can be detected expediently and accurately.

Furthermore, when standards established in known steps of quantity areused, the quantitation of the mutant p51 in the test sample can be madeat the same time by comparing the peak areas of the standards with thepeak area of the amplification product of p51 DNA in the test sample inthe PCR amplification step using the mutant primer set mentioned above.The primer set, standards, PCR-SSCP analysis and detection means can beliberally modified by those skilled in the art and the present inventionencompasses such modifications, of course, inasmuch as the sequences ofthe wild type p51 gene and mutant p51 genes are employed.

The above assay technology according to the present invention is nowdescribed more specifically. To begin with, the DNA is extracted fromthe serum of a cancer patient by the routine procedure such as alkali oracid treatment. Then, a primer set comprising a minus chain partialsequence of a defined length consisting in a part of the nucleotidesequence (145-1488) shown under SEQ ID NO:1 and a plus chain partialsequence of a defined length consisting in a part of thefluorescent-labeled nucleotide sequence (145-1488) as well as aheat-resistant DNA polymerase are caused to act on the DNA solutionobtained above to amplify the labeled DNA fragment.

On the other hand, one or a plurality of DNA fragments containing amutant sequence chemically synthesized according to the p51 sitemutation information obtained from, for example, a cancer patient arerespectively integrated in plasmid vectors and E. coli is transformed.After mass culture and purification, the purified recombinant plasmidsare used to prepare e.g. 10³ copy, 10⁴ copy, 10⁵ copy, 10⁶ copy, 10⁷copy and 10⁸ copy standards. Said primer set comprising a minus chainpartial sequence consisting in a defined partial sequence of saidnucleotide sequence (145-1488) and a plus chain partial sequenceconsisting in a defined partial sequence of the fluorescent-labelednucleotide sequence (145-1488) as well as a heat-resistant DNApolymerase to amply the labeled DNA fragment. The solution of DNAamplified above is heated at about 95° C. for about 5 minutes, thenimmediately cooled on ice, and a SSCP analysis is performed using anautomatic sequencer, such as ALF Automatic Sequencer (Pharmacia),whereby the fluorescent peak can be detected. Phoresis in this SSCPanalysis is performed preferably at about 30° C.±1° C.

The peak (mobility) of the DNA obtained from the patient's serum iscompared with the peaks (mobilities) of the standards and the peak inagreement with a standard is ascertained from the migration time. Inthis manner, the type (kind) of mutation of the patient p51 can beascertained. Moreover, by calculating the peak areas of standards andconstructing a standard curve, the p51DNA can be quantitated from thecalculated peak area of the patient's DNA.

(9) Method of Detecting Mutation of the p51 Gene and Various AssayMethods

The present invention, therefore, provides an expedient test protocolfor the concurrent detection and quantitation of mutation in a givenregion of p51 DNA in the test sample through the above measurement.

The assay method of this invention can be carried out conveniently byutilizing a reagent kit for detecting the wild type p51 gene and mutantp51 gene in a sample.

Therefore, the present invention further provides a reagent kit fordetection of wild type p51 and mutant p51 characterized by itscomprising said wild type p51 DNA fragment and mutant p51 DNA fragment.

This reagent kit, inasmuch as it contains a DNA fragment capable ofhybridizing with a part or the whole of the nucleotide sequence(145-1488) shown under SEQ ID NO:2 or its complementary oligonucleotidesequence or a DNA fragment capable of hybridizing with a part or thewhole of a mutant sequence of the nucleotide sequence (145-1488) or anucleotide sequence complementary to said sequence, may contain othercomponents such as a labeling agent, reagents essential to PCR (e.g.TaqDNA polymerase, deoxynucleotide triphosphate, primers, etc.). In lieuof the nucleotide sequence (145-1488) shown under SEQ ID NO:2, thenucleotide sequence (145-2067) shown under SEQ ID NO:5 can be used.

As the labeling agent, a radio isotope or a chemical modifier such as afluorescent substance can be employed, although the DNA fragment itselfmay have been conjugated with the labeling agent in advance. Moreover,this reagent kit may further comprise a suitable reaction diluent,standard antibody, buffer, washing solution, reaction stopper, etc. forconvenience in carrying out an assay.

The present invention further provides a method of diagnosis using saidassay technique and a diagnostic agent and a diagnostic kit for use insaid method of diagnosis.

Further, by direct or indirect sequencing of the mutant p51 sequenceobtained from the test sample by the above procedure, it is possible todiscover novel p51-related genes having high homology to the wild typep51.

The present invention, therefore, further provides a method of screeningfor human p51-related genes in test samples through the above-describedassay and sequencing of mutant p51 DNA in such test samples.

Furthermore, the wild type p51 and/or mutant p51 can be identified bysynthesizing the protein encoded by the human p51A gene of SEQ ID NO:1or the protein corresponding to the amino acid sequence derived from thesequence of SEQ ID NO:1 by deletion, substitution or addition of one ora plurality of amino acids or a partial sequence thereof, orsynthesizing the antibody against any of such proteins. Furthermore, inlieu of the protein encoded by said human p51A gene, the protein encodedby the human p51B gene shown under SEQ ID NO:4 can be used.

Therefore, the present invention provides a method for assay ofantibodies against wild type p51 and/or mutant p51 and for assay of theantigen. By this assay method, the degree of neoplastic disturbance orthe malignancy of a malignant tumor can be estimated based on the changein the wild type p51 protein. The change mentioned above can bedetermined by p51 sequence analysis by said routine technology but morepreferably the change in the p51 protein or the presence or absence ofthe p51 protein is detected using an antibody (a polyclonal antibody ora monoclonal antibody). A specific example of the assay method of theinvention is as follows. With the p51 antibody, the p51 protein can beimmunoprecipitated from a solution containing a human biologicalmaterial isolated from a human being, such as blood or serum, and theantibody can be reacted with the p51 protein on a polyacrylamide gelWestern blot or immunoblot. Moreover, with the p51 antibody, the p51protein in a paraffin section or frozen tissue section can be detectedby an immunohistochemical technique. The technology for antibodyproduction and purification is well known in the art and the knowntechniques can be selectively employed.

The preferred specific modes of practicing the method of detecting thewild type p51 or a mutant thereof include enzyme-linked immunosorbentassay (ELISA) inclusive of the sandwich technique using a monoclonalantibody and/or a monoclonal antibody, radioimmunoassay (RIA),immunoradiomatrix assay(?) (IRMA) and immunoenzymematrix assay(?)(IEMA).

Furthermore, in accordance with the present invention, it is alsopossible to provide a cell membrane fraction having p51-binding activityfor the p51 protein or the p51 receptor present on the cell surface. Toacquire said p51 receptor, the labeled p51 protein is conjugated in abiological sample containing the cell membrane fraction, the resultingp51 conjugate is isolated by extraction and purified, and the amino acidsequence of the isolate is determined. The acquisition and sequencing ofthis p51 receptor protein fall within the expertise of one skilled inthe art.

(10) Application to Drug Screening

The present invention can be applied to the screening for compounds (p51receptor reaction products: the compound may be a low molecularcompound, a high molecular compound, a protein, a protein fragment, anantigen, an antibody or the like) by using the p51 receptor polypeptideor a binding fragment thereof for the screening of various drugs.Preferably, the p51 receptor protein is utilized. The p51 receptorpolypeptide or its fragment for use in such a screening test may beimmobilized on a solid support or used in the form of a suspension in afluid carried to the cell surface. To mention an example of drugscreening, host eucaryotic or procaryotic cells transformed stably witha recombinant polypeptide and expressing the polypeptide or its fragmentcan be utilized, preferably in a competitive binding assay. Moreover,such cells in the free or immobilized state can be used in a standardbinding assay. More preferably, the formation of a complex between thep51 receptor polypeptide or its fragment and a test substance isquantitated to detect the degree of inhibition of said formation of acomplex between the p51 receptor polypeptide or fragment and the p51polypeptide or fragment by the test substance is detected, whereby thescreening for a compound can be accomplished.

Thus, the present invention provides a method of drug screeningcharacterized by contacting such a substance with the p51 receptorpolypeptide or a fragment thereof by a per se known technique and, then,detecting the presence of a complex between said substance and said p51receptor polypeptide or fragment or the presence of a complex betweensaid p51 receptor polypeptide or fragment and a ligand. Further, the p51receptor activity is measured to see whether said substance mayantagonize the p51 receptor to exhibit the p51 activities definedhereinbefore, for example the activity to modify the cell cycle ormodulate the induction of apoptosis. Specifically, in carrying out sucha competitive binding assay, the p51 receptor polypeptide or itsfragment is labeled. The free p51 receptor polypeptide or fragment isseparated from the protein-protein complex. Then the amount of the freelabel (non-complex-forming) can be a measure of the binding between thefactor to be assayed and the p51 receptor or inhibition of the p51receptor-p51 polypeptide binding. The small peptide (pseudopeptide) ofthe p51 polypeptide is thus analyzed, and the one having p51 receptorinhibitory activity is determined.

Another drug screening method of the present invention is a method ofscreening for compounds having an adequate binding affinity for the p51receptor polypeptide. Briefly, a large number of different peptide testcompounds are synthesized on solid supports such as plastic pins orother surfaces. Then, the peptide test compounds are reacted with thep51 receptor polypeptide, followed by washing. Then, the reacted andbound p51 receptor polypeptide is detected by a per se known technique[PCT: WO84-03564]. The purified p51 receptor can be directly coated on aplate for use in said drug screening. However, the p51 receptorpolypeptide can be immobilized on a solid phase by antibodysupplementation using a non-neutralizing antibody against thepolypeptide. Furthermore, the present invention is directed to acompetitive drug screening assay in which a neutralizing antibodyspecifically binding the p51 receptor polypeptide and a test compoundare caused to compete with each other for the binding to the p51receptor polypeptide or fragment. By this competitive assay using theantibody, the presence of peptides having one or more epitopes orantigenic determinant sites of the p51 receptor polypeptide can also bedetected.

Referring, further, to drug screening, a still another method comprisesthe use of a host eucaryotic cell line or cells containing anonfunctional p51 gene. Thus, after the host cell line or cells areallowed to grow in the presence of a drug compound for a predeterminedtime, the proliferation rate of the host cells is measured to seewhether the compound may modulate apoptosis or the cell cycle. As ameans for measuring the proliferation rate, it is possible to measurethe biological activity of the p51 receptor.

Moreover, in accordance with the present invention, for the purpose ofdeveloping the more active or stable p51 polypeptide derivatives ordrugs which will potentiate or interfere with the function of the p51polypeptide in vivo, various interactive biologically activepolypeptides or structural analogs, e.g. p51 agonists, p51 antagonists,p51 inhibitors, etc., can be constructed. Such structural analogs can becharacterized by, for example, analyzing the three-dimensionalstructures of complexes between p51 and other proteins by X-raycrystallography, computer modeling or a combination of such techniques.Furthermore, the information on structural analogs can also be generatedby protein modeling based on the structures of homologous proteins.

The method of obtaining the still more active or stable p51 polypeptidederivatives may for example involve an alanine scan analysis. In thismethod, Ala is substituted for each amino acid residue and the effect ofsubstitution on peptide activity is determined. Each amino acid residuein a peptide is thus analyzed and the region or regions of importance tothe activity or stability of the peptide are determined. By using thismethod, the more active or stable p51 derivatives can be designed.

It is also possible to isolate the target-specific antibody selected bya functional assay and analyze its crystal structure. As a rule, apharmacore providing the basis for further drug design can be obtainedby this approach. It is possible to identify or isolate a peptide from achemically or biologically constructed peptide bank by causing formationof an anti-ideotype antibody against a functional pharmacoactiveantibody. Therefore, the selected peptide is also expected to serve as apharmacore.

In this manner, drugs having improved p51 activity or stability or drugsacting as inhibitors, agonists or antagonists of p51 activity can bedesigned and developed.

In accordance with the cloned p51 sequence, a sufficient amount of p51polypeptide can be procured and submitted to X-ray crystallographic andother analytical research. Furthermore, the p51 protein having the aminoacid sequence of SEQ ID NO:1 as provided by the present inventionenables establishment of a computer modeling program which may take theplace of X-ray crystallography or supplement the latter technique.

Furthermore, by constructing a human p51 gene-bearing knockout mouse(mutant mouse) in accordance with the present invention, it is possibleto detect which sites of the human p51 gene sequence will influence saidvarious p51 activities in vivo, that is to say what functions the p51gene product and mutant p51 gene products will have in vivo.

This is a technology to intentionally modify the genetic information oforganisms by utilizing homologous recombinations of genes, and themethod using mouse embryonic stem cells (ES cells) can be mentioned asan example [Capeccchi, M. R. Science, 244, 1288-1292 (1989)].

The method of constructing such mutant mice as above is well known tothose skilled in the art, and by applying the human wild-type p51 geneor mutant p51 gene of the present invention to the above technology asmodified (Noda, T. (ed.), Experimental Medicine, Supplemental Issue,14(20) (1996), Yōdo-sha), mutant mice can be easily established.Therefore, by utilizing the above technology, drugs having improved p51activity or stability or drugs acting as inhibitors, agonists orantagonists of p51 activity can be designed and developed.

The present invention comprises the following.

-   1. A method of inhibiting tumorigenesis which comprises transferring    the p51 gene to tumor cells.-   2. A method of inhibiting tumorigenesis which comprises transferring    the p51 protein to tumor cells.-   3. A pharmaceutical composition comprising the p51 gene or an    equivalent thereof and a pharmaceutically acceptable carrier.-   4. A pharmaceutical composition comprising the p51 protein or an    equivalent thereof and a pharmaceutical acceptable carrier.-   5. A drug for gene therapy which comprises the p51 gene or an    equivalent thereof as an active ingredient.-   6. A cancer diagnostic reagent comprising the p51 gene or an    equivalent thereof.-   7. A cancer diagnostic reagent comprising the p51 protein or an    equivalent thereof.-   8. A method of screening for p51- or p53-related genes which    comprises using the p51 gene or an equivalent thereof.-   9. A method of screening for inhibitors of cell tumorigenesis which    comprises using the p51 gene or an equivalent thereof.-   10. A method of screening for p51 gene inducers and/or inhibitors    which comprises using the p51 gene or an equivalent thereof.-   11. Use of a p51 gene inducer and/or inhibitor selected by the above    screening in the therapy of diseases arising from p51 gene    expression abnormality.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples and experimental examples are furtherillustrative of the present invention. It should, however, be understoodthat the scope of the invention is by no means restricted by theseexamples and experimental examples.

EXAMPLE 1 Isolation of the Human p51 Gene

(1) Cloning and DNA Sequencing of the Human p51 Gene

(a) The present inventors carried out a PCR amplification using thefollowing p73-F1 sense primer and p73-R1 antisense primer and then asecond amplification by a nested PCR using the following p73-F2 senseprimer and p73-R2 antisense primer.

p73-F1: (SEQ ID NO:12) 5′-TA(CGT)GCA(CGT)AAA(G)ACA(CGT)TGC(T)CC-3′p73-R1: (SEQ ID NO:13) 3′-TGC(T)GCA(CGT)TGC(T)CCA(CGT)GGA(CGT)A(C)G-5′p73-F2: (SEQ ID NO:14) 5′-TA(CGT)ATA(CT)A(C)GA(CGT)GTA(CGT)GAA(G)GG-3′p73-R2: (SEQ ID NO:15) 3′-ATGAAC(T)A(C)GA(CGT)A(C)GA(CGT)CCA(CGT)AT-5′

More specifically, from the human skeletal muscle polyA+RNA (Clontech),the cDNA was synthesized using a random primer and an oligo dT primer.Then, a cDNA library consisting of about 10⁷ plaques as constructedusing λ ZipLox (Gibco BRL) as the vector was amplified and the DNA wasextracted. Using 0.2 μg of the cDNA as the template and said p73-F1 andp73-R1 as primers, an amplification reaction was carried out in 25cycles of 94° C., 30 sec., 45° C., 30 sec. and 72° C., 30 sec inaccordance with the Tag Polymerase (Gibco-BRL) manual. Then, using 1/100of the amplification product as the template and said p73-F2 and p73-R2as primers, a further amplification was carried out under the sameconditions.

Since a band of 172 bp as deduced from the structure of the p53 gene wasobtained, a restriction enzyme cleavage map of the band was prepared. Asa result, the presence of a gene other than the p53 gene was detected.This band was subcloned in pGEM7 (Promega) and using ABI377 AutomaticSequencer (ABI), the nucleotide sequence was determined in the routinemanner. As a result, it was found to be a DNA fragment derived from anovel gene which resembles the p53 gene but has a different novelnucleotide sequence.

Separately, a similar analysis was carried out using cDNA librariesderived from other organs (e.g. brain). As a result, a DNA fragmentderived from a further different novel gene resembling the p53 gene wasdetected but it was found to be a fragment derived from the p73 gene.

This subcloned DNA fragment was excised and using the BcaBest labelingkit (Takara), a labeled probe was constructed. The plaque hybridizationassay of an unamplified library of 2.4×10⁶ plaques as constructed usingthe oligo dt primer alone in otherwise the same manner as in theconstruction of said cDNA library gave 8 positive clones. Since λ ZipLoxcan be easily converted to a plasmid using the Cre-LoxP system,sequencing of the plasmid obtained by conversion was carried out in theroutine manner using LICOR's automatic sequencer and ABI377 automaticsequencer (ABI).

Then, between the nucleotide sequence of the gene obtained and thenucleotide sequences of the p53 and p73 genes, a homology search wasmade with FASTA Program using GCG software (Wisconsin SequencingPackage, Genetics Computer Group) [Person, W. R. and Lipman, D. J.,Proc. Natl. Acad. Sci. U.S.A., 85, 1435-1441 (1988)].

As the result of said homology search, two of the clones selected by theabove method and sequenced were found to have high homology with respectto the p53 gene and p73 gene. The molecular masses calculated from thededuced amino acids encoded by the gene sequence of these 2 clones were50,894 Da and about 71,900 Da, respectively. The present inventors namedthese clones p51A and p51B, respectively.

The full-length nucleotide sequence of the gene (p51A gene) possessed bythe p51A clone obtained as above is shown under SEQ ID NO:2 and thefull-length nucleotide sequence of the gene (p51B gene) possessed by thep51B clone is shown under SEQ ID NO:5.

As shown under SEQ ID NO:2, the p51A clone was found to have a genehaving a nucleotide sequence (1344 nucleotides) coding for the aminoacid sequence (448 amino acids) of SEQ ID NO:1 and having an openreading frame in the 145 th˜1488 th position. Moreover, the deducedamino acid sequence encoded by the nucleotide sequence of the genepossessed by this clone had a transcriptional activation domain in the 1st˜59 th position, the DNA binding domain in the 142 nd˜321 st position,and the oligomerization domain in the 359 th˜397 th position.

On the other hand, as shown under SEQ ID NO:5, the p51B clone was foundto have a gene having a nucleotide sequence (1923 nucleotides) codingfor the amino acid sequence (641 amino acids) of SEQ ID NO:4 and havingan open reading frame in the 145 th˜2067 th position. Moreover, thededuced amino acid sequence encoded by the nucleotide sequence of thegene possessed by this clone had a transcriptional activation domain inthe 1 st˜59 th position, a DNA binding domain in the 142 nd˜321 stposition, and an oligomerization position in the 353 rd˜397 th position.Furthermore, this sequence was found to have an additional sequence (SAMdomain) in the C-terminal region and the 353 rd˜641 st region inclusiveof this additional sequence could be regarded as an oligomerizationdomain in a broad sense of the term.

The amino acid sequence encoded by the p51A gene of the invention wascompared with the amino acid sequences of the p53 protein and p73βprotein for homology comparison among the three sequences (FIG. 2). Inthe diagram, the amino acids common to the 3 sequences are boxed.

FIG. 1 is a schematic diagram showing features of the structural domainsof the p51A protein along with those of the p53 protein and p73βprotein. In the diagram, “TA” represents a transcriptional activationdomain, “DNA binding” represents a DNA-binding domain, and “Oligo”represents an origomerization domain. The structural features of the p51protein and p73β protein were deduced from the structural features ofthe p53 protein.

As a result, the homology of the deduced amino acid sequences of thep51A protein, p53 protein and p73β protein in each of full-lengthsequence, transcriptional activation domain, DNA-binding domain, andoligomerization domain was respectively as follows: between p51A proteinand p53 protein, 36%, 22%, 60% and 37%, respectively; between p51Aprotein and p73 protein, 42%, 30%, 87% and 65%, respectively; andbetween p53 protein and p73 protein, 28%, 27%, 63% and 83%, respectively(Table 1).

Moreover, although the 448 residue-structure of the p51A protein wasshorter than the 636 residue-structure of the p73α protein, the totalstructure of the p51A protein resembled the p73 protein with theC-terminal region split off.

These results indicated that although the putative amino acid sequenceof the p51A protein resembles the deduced sequences of both the p53protein and p73β protein, its homology to the amino acid sequence of thep73β protein is higher than its homology to the p53 protein and that thehomology between p51A protein and p73β protein is higher than thehomology between p53 protein and p73β protein in the regions other thanthe oligomerization domain. Furthermore, between p51A protein and p73βprotein, a homology was found in the region where no homology was foundbetween p53 protein and p73β protein or between p53 protein and p51Aprotein. These results suggest that, on the amino acid sequence level,the p51A protein can be said to be closer to the p73β protein than tothe p53 protein.

Similarly, the amino acid sequence encoded by the p51B gene of theinvention was compared with the amino acid sequence of the p73α proteinfor homology comparison (FIG. 3). In the diagram, the amino acids commonto the two sequences are boxed.

FIG. 4 is a schematic diagram showing features of the structural domainsof splicing variants encoded by the p51 (A and B) genes along with thoseof the p73 proteins (α and β). Whereas the divergence point between p51Aprotein and p51B protein begins at intron 10 and the divergence pointbetween p73α protein and p73β protein begins at intron 13.

EXAMPLE 2 Confirmation of p51mRNA expression in Normal Human Tissue

(1) Northern Blot Analysis

Expression of p51mRNA in normal human tissue was assessed by Northernblotting using, as the probe, a human cDNA clone labeled by the randomoligonucleotide priming method.

Northern blot analysis was carried out using Human Multiple TissueNorthern Blot (Clontech, Palo Alto, Calif., U.S.A.) in accordance withthe product manual.

Thus, the EcoRI fragment (600 bp: corresponding to the 5′ end of cDNA)of a PCR amplification product of the DNA clone obtained in Example 1was labeled with [³²p]-dCTP (Random Primed DNA Labeling Kit, BoehringerMannheim) for use as a probe.

Blotting was made using ExpressHyb Hybridization Solution (Clontech)under the conditions directed in the user manual, and detection was madeusing BAS2000 (FUJI).

The results are shown in FIG. 5 and FIG. 6.

FIG. 5 shows the result of Northern hybridization carried out with thefilter purchased from Clontech, and FIG. 6 shows the result of Northernhybridization carried out with a filter constructed by the presentinventors using the RNA purchased from Clontech. FIG. 5 shows theelectrophoretogram with 2 μg poly A-RNA added for each lane, and FIG. 6is the electrophoretogram with 0.5 μg poly A+RNA added for each lane.

The lanes in FIG. 5 represent the result for 1: heart, 2: brain, 3:placenta, 4: lung, 5: liver, 6: skeletal muscle, 7: spleen, and 8:pancreas. The lanes in FIG. 6 represent the result for 1: mammary gland,2: prostate, 3: salivary gland, 4: stomach, 5: thymus, 6: thyroid, 7:trachea, and 8: uterus.

It was found that the distribution of expression of the mRNA (4.4 kb) ofthe gene named “human p51 gene” according to the present invention wasrather confined in contrast to the ubiquitous expression of p53 mRNA,with the expression level being highest in skeletal muscle, seconded byplacenta, and decreased progressively in placenta, trachea, mammarygland, prostate, salivary gland, thymus, uterus, stomach, lung, brainand heart in the order mentioned. In other tissues (e.g. adrenal gland,small intestine, spinal cord, spleen), no expression of p51mRNA could bedetected.

The expression pattern of the p73 gene is also tissue-restricted.However, it was found that while the expression of p51 gene overlappedthe expression of the p73 gene (expression in the same tissue), thedistribution of expression was broader than the distribution ofexpression of the p73 gene.

The above difference in expression tissue distribution among the humanp51 gene, p53 gene and p73 gene suggested that notwithstanding theresemblance in biological activity among these genes, they aredissimilar in function depending on tissues in vivo.

Further research also revealed that, in various human tissues, thep51mRNA, as in the case of p73 protein, exists in selectively splicedforms (alternative splicing variants), namely a short form encoding thep51A protein and a long form encoding the p51B protein. The latter longform encoding p51B was found to have homology to the factor named ketwhich had been accidentally discovered in a search for the glutamatereceptor of the tongue. The 3 kb mRNA, which is a main transcript inskeletal muscle, was the most abundant mRNA observed in all the tissuesinvestigated. The short-form cDNA clone was suspected to be derived fromthis transcript. Interestingly, in contrast to the mRNA observed innormal tissues, this short-form (p51A) of p51mRNA was found to have beenexpressed in many tumor cell lines.

FIG. 4 is a schematic diagram comparing the structures of the respectivealternative splicing variants of the p51 protein and p73 protein. Thisp51BmRNA encoded a protein having a molecular mass (calculated) similarto that of p73α.

Functional differences between p51A and p51B remain unknown.

EXAMPLE 3 Chromosome Mapping of the p51 Gene

Using a radiation hybrid panel (GeneBridge 4 Radiation Hybrid Panel;Research Genetics), the p51 gene was mapped on the human chromosome. Asa result, the p51 gene was localized in the 3q28-ter region between themarkers AFBM327YD9 and WI-1189 (5.66 cR from the former marker).

EXAMPLE 4 Mutation of p51 in Various Human Cancer Cell Lines and HumanTumors

The most intriguing question about the p51 gene is the question ofwhether the features of the p53 tumor suppressor gene are shared by thep51 gene as well as the relationship of a mutation of the present genewith the morphogenesis of a human tumor.

Therefore, using various tumor cell lines, a search was made for thepresence or absence of mutation of the p51 gene. For this search, themethod for functional analysis of separated alleles in yeasts (FASAY),which was previously used by the present inventors in the identificationand characterization of p53 mutation, was used [Ishioka et al., Nat.Genet. 5. 124-129 (1933)].

A complementary full-length DNA fragment coding for the human p51A genewas amplified by the same PCR method as used in the determinationdescribed hereinbefore to acquire the nucleotide sequence of theamplification product covering the full-length sequence encoding thep51A gene and this nucleotide sequence was determined by directsequencing to detect the presence or absence of a mutation.

Tumor cells were respectively cultured in Dulbecco's Modified EssentialMedia supplemented with 10% fetal calf serum in a 5% CO₂ environment.Since all the p51AcDNA clones could amplify the p53cDNA in the previousanalysis, the quality of cDNAs of cell lines was guaranteed.

Of 102 cell lines, 67 lines analyzed were capable of amplifying the p51ADNA fragment. The nucleotide sequence was determined by directsequencing for 35 of the above cell lines.

Mutations were found in two cell lines, namely Ho-1-u-1 (JCRB0828),which is a head-and-neck cancer cell line, and SKG-IIIa (JCRB0611),which is a cervical cancer cell line.

The mutation was Ser¹⁴⁵→Leu in the former and Gin¹⁶⁵→Leu in the latter.With regard to the p53 protein, it was likely that the normal functionof the p53 protein had been defected by mutation in the former and byhuman papilloma virus infection in the latter. Moreover, in the mRNAsderived from tumor cells, various splicing variants were noted.

Referring to human primary cancers, the nucleotide sequences of the DNAamplification products obtained by SSCP and RT-PCR techniques weredetermined by direct sequencing in search for p51A gene mutation. In atotal population of 66 human tumor cases, namely 8 neuroblastoma cases,8 colon cancer cases, 8 breast cancer cases, 8 lung cancer cases, 8brain tumor cases, 8 esophageal cancer cases, 8 hepatocellular cancercases, 6 pancreatic cancer cases, and 4 renal cancer cases, a mutationof Ala¹⁴⁸→Pro was detected in one lung cancer case.

The analysis of the above 3 cases was invariably an analysis of cDNA andit was clear that the expression originated from a single chromosomallocus.

EXPERIMENTAL EXAMPLE 1 Suppression of Colony Formation by p51Transformation

The p53 protein has an ability to block cells in the G1 phase or induceapoptosis.

To investigate the colony formation inhibitory activity of the p51protein of the invention, the SAOS2 osteosarcoma cell line (accessionnumber: ATCC HTB85) was co-transfected with a puromycin-resistantexpression plasmid (pBABEpuro: Morgenstern J. Nuc. Acids Ru, 18, 3587,1990) as well as a p51A expression construct, an HA-labeled p51Aexpression construct (HA-labeled ATGTATCCATATGATGTTCCAGATTATGCT (SEQ IDNO:16), which codes for the amino acid sequence MYPYDVPDYA (SEQ ID NO:17)), a p53 expression construct, and a vector and the colony-formingability was evaluated.

The above expression vectors were constructed by cloning the codingregion fragment of p51A DNA (2816 nucleotides; in SEQ ID NO:2,oligonucleotide numbers 1˜2816), the fragment prepared by adding the HAtag to the p51A cDNA, and the coding region fragment of p53cDNA (1698nucleotides; nucleotide numbers 62˜1760), respectively. Then, theosteosarcoma cell line SAOS2 was cultured in Dulbeccos's ModifiedEssential Medium supplemented with 10% fetal calf serum in a 5% CO₂environment. A 6 cm dish was seeded with the above SAOS2 cells (1×0⁶cells/dish) and, after 24 hours, the cells were transfected with awild-type p51 expression vector containing the p51A cDNA chain(pRcCMV/p51A). Similar transformations were carried out using theHA-tagged p51A cDNA, the wild-type p53 gene and, as control, the pRcCMVexpression vector alone.

Using Mammalian Transfection Kit (Stratagene), 1 μg of pBABEpuro wasintroduced into the cells. The resulting cells were fixed and stainedwith Crystal Violet. The stained colonies were photographed. Eachtransfection was carried out twice for analyzing colony formation.

As a result, significant decreases in the number of colonies wereobserved in the culture dishes of p53 gene-transfected cells and p51gene-transfected cells. In contrast, in the culture dish of cellstreated with the vector alone, growth of a large number of colonies wasobserved. The p51 gene was thus found to have an ability to suppresscolony formation but this ability was slightly poor as compared with theability of the p53 gene. On the other hand, the HA-tagged p51 geneshowed a colony formation-suppressing ability comparable to that of thep53 gene (FIG. 7).

EXPERIMENTAL EXAMPLE 2 Test of the Transcriptional Activation Functionof the p51 Protein

Since the activity of the p53 protein to arrest cell growth in G1 phaseor induce apoptosis was dependent on the transcriptional activationfunction of the p53 protein, a test was carried out to see whether thep51 protein would exhibit such activity.

Downstream of the Waf1 promoter, which is known to be controlled by p53transcriptional activation function, and RGC (ribosomal gene cluster)sequence, a luciferase reporter plasmid as well as a p51A geneexpression construct was introduced by the method described in Example5. Specifically, SAOS2 cells were co-transfected with said luciferasereporter plasmid and the p51A expression vector, p53 expression vectoror control vector and the luciferase activity of the lysate obtainedfrom the resulting transformant was assayer. The luciferase activity wascalculated with a dual luciferase reporter assay system (ProMega) takingthe transfection efficiency into consideration.

FIG. 8 is a schematic diagram showing the reporter construct used in theexperiment. Shown in the diagram are 3 luciferase gene constructs eachlinked downstream of various p21WAF1 promoters. In the diagram, “WAF-1promoter Iuc” represents a wild-type p21WAF1 promoter constructretaining the two p53 control elements; “del 1” represents the constructdeprived of the upstream one of said elements; and “del 2” representsthe construct deprived of both of said elements.

The results are shown in FIG. 9 and FIG. 10. Relative activity on theordinate represents the luciferase activity calculated by using the dualluciferase reporter assay system taking the transfection efficiency intoconsideration.

FIG. 9 shows the transactivation activity found when the p51 expressionplasmid (p51A), the p53 expression plasmid (p53) or the vector (Rc/CMV)only, which has been linked to each of the various reporter constructsshown in FIG. 8, was introduced into SAOS2 cells. The results showedthat like the p53 protein, the p51 protein has activity to induce thenumber-dependent expression of the p53 reactive sequence.

FIG. 10 shows the results of a similar experiment using the p51Aexpression plasmid (p51A), the HA-tagged p51A expression plasmid(Hap51A), p53 expression plasmid (p53) or the vector (RcCMV), which hasbeen linked to the PGC reporter construct which has been experimentallydemonstrated to have p53 reactivity. As in the experiment shown in FIG.9, the above results indicated that both p51A and HAp51A, like p53, haveactivity to induce the number-dependent expression of the p53 reactivesequence. The weak activity found when the p51A expression plasmid wasused may be attributed to the fact that since this plasmid was builtinto the expression vector with the leader sequence retained, the amountof expression was small.

When the leader sequence was removed in a later experiment, the p51Aprotein showed a stronger expression-inducing action than the p53protein and, in said colony formation-inhibition assay, too, thisprotein was found to have strong activity.

It is apparent from the above results that the p51 protein had theability to induce transcription through its transcriptional regulationdomain. The finding that the transcriptional activity was lost oninduced mutation of this element suggests that the p51 protein alsoutilizes the same recognition sequence as does the p53 protein.

Then, it was inquired whether the same transcription relation holds truein vivo, too. A p51A gene expression construct having an HA-taggedepitope was introduced into SAOS2 cells over a short time. The findingof the uptake of the p51A gene by cells indicated that p51A is localizedin the nucleus and all the cells were found to elevate the level ofp21Waf1. This indicates that the p51 protein is also capable of inducingp21Waf1 which is known to be controlled by the p53 protein.

EXPERIMENTAL EXAMPLE 3 p51 Gene Mutation in Tumors in situ

The mutation of the p51 gene was investigated in the in situ cancercells of 66 patients (neuroblastoma, 8 cases; colon cancer, 8 cases;breast cancer, 8 cases; lung cancer, 8 cases; brain tumor, 8 cases;esophagus cancer, 8 cases, hepatocytoma, 8 cases; pancreatic cancer, 6cases, and renal cancer, 4 cases) by the reverse transcription PCRsingle-stranded structure polymorphism (RT-PCR-SSCP) method and DNAsequencing method.

(1) Preparation of RNA

Fresh tumor samples were surgically isolated, immediately frozen andstored at −80° C. until used. The RNA was extracted by the methoddescribed in the report of Nakagawa et al. [Nakagawa, A., et al., N.Engl. J. Med., 328, 847-854 (1993)].

(2) RT-PCR-SSCP and DNA Sequencing

The total RNA, 5 μg, was transcribed on cDNA using Superscript IIreverse transcriptase (Gibco-BRL) and random primers. The 20th cDNA ofthis reaction product was used for PCR amplification. PCR-SSCP wasperformed in accordance with the method of Mashiyama et al. [MashiyamaS. et al., Oncogene, 6, 1313-1318 (1991)]. Specifically, the PCR productwas amplified using 3 primers for p51A cDNA.

The nucleotide sequences of primers used for PCR are as follows.

p51-F1: 5′-AAAGAAAGTTATTACCGATG-3′ (SEQ ID NO:18) p51-R1:5′-CGCGTGGTCTGTGTTATAGG-3′ (SEQ ID NO:19) p51-F2:5′-CATGGACCAGCAGATTCAGA-3′ (SEQ ID NO:20) p51-R2:5′-CATCACCTTGATCTGGATG-3′ (SEQ ID NO:21) p51-F3:5′-CCACCTGGACGTATTCCACT-3′ (SEQ ID NO:22) p51-R3:5′-TGGCTCATAAGGTACCAG-3′ (SEQ ID NO:23) p51-F4:5′-CATGAGCTGAGCCGTGAAT-3′ (SEQ ID NO:24) p51-R4:5′-TATCTTCATCCGCCTTCCTG-3′ (SEQ ID NO:25) p51-F5:5′-ATGAACCGCCGTCCAATT-3′ (SEQ ID NO:26) p51-R5:5′-GTGCTGAGGAAGGTACTGCA-3′ (SEQ ID NO:27) p51-F6:5′-TGAAGATCAAAGAGTCCCTG-3′ (SEQ ID NO:28) p51-R6:5′-CTAGTGGCTTTGTGCCTTTG-3′ (SEQ ID NO:29)

Then, the 32PdCTP was diluted 1:10 with loading buffer. After 5 minutesof further denaturing at 98° C., the separation was carried out on 5%glycerol/5% polyacrylamide gel at 200 volts at room temperature for12˜14 hours. After the electrophoresis, the gel was dried and exposedagainst X-ray film overnight so that the migration bands would bedefinitely visible. To confirm the presence or absence of mutation, thePCR product was subcloned into the pGEM-T Easy Vector (Promega),followed by sequencing with ABI377 DNA sequencer.

As a result, in the lung cancer tissue belonging to the type of highlydifferentiated squamous cell cancer, an amino acid substitution pointmutation (Ala¹⁴⁸→Pro) was found in the deduced DNA binding region of thep51A protein. This tumor showed paratracheal lyphnode metastasis andpleural invasion. Since all of randomly selected 5 clones had the samemutation, the p51 gene possessed by this tumor cell was suggested to bea single allele or have been expressed mono-allelically.

EXPERIMENTAL EXAMPLE 4 Induction of Apoptosis by p51cDNA Introduction

It was explored whether, like the p53 protein, the p51 protein wouldinduce cell apoptosis.

The apoptosis induction test with the p51 protein was carried out by themethod of the present inventors, namely the method which, as mentionedabove, comprises the use of a transgenic mouse erythroleukemia cell line(1-2-3 cell line) which presents with typical features of apoptosis whencultured at 32° C. [Kato, M. V., et al., Int. J. Oncol., 9, 269 (1996)].

This mouse erythroleukemia cell line (1-2-3 cell line) was establishedfrom the erythroleukemia derived from Friend spleen focus forming virusgp55 gene-transgenic mice [Xu et al., Jpn. J. Cancer Res. 86, 284-291(1995); Kato et al., Int. J. Oncol. 9 269-277] and is a cell strainwhich expresses only a temperature-sensitive (ts) mutant p53 protein(Ala1353Val: point mutation). This ts-mutant p53 protein is localized inthe cytoplasm at 37° C., which is a usual culture temperature, and,therefore, does not exhibit the control function of the p53 moleculewhich is intrinsically discharged in the nucleus but at 32° C. itmigrates into the nucleus so that the p53 activity is induced [Levine,A. J. et al., Nature 351, 453-456 (1991)]. It has been already reportedthat, in this cell line, slow apoptosis is induced at 32° C.

The 1-2-3 cells were cultured in RPMI 1640 medium supplemented with 10%fetal calf serum in a 5% CO₂ environment. Then, using pRc/CMV as theexpression vector, the p51A gene was introduced into the above cells.The cells were then cultured in a selective medium, and using neomycinresistance (Neo^(r)) as the test, G418-resistant cells were selected. Anapoptosis induction study was then carried out in the p51A-expressingcells.

Thus, two strains of p51A-transfected 1-2-3 cells (hereinafter referredto as “1C1 cells” and “4B1 cells”) as transfected with the p51Agent-harboring expression vector (pRcCMV/p51A) and, as control, 1-2-3cells transformed with the vector alone and not containing the p51A genewere respectively seeded on 10 cm (dia.) plates at a concentration of1×10⁵/ml and cultured at 2 alternative temperatures of 37° C. and 32° C.for 24 hours. The cells were then harvested and treated with proteinaseK and Rnase A to prepare DNA samples. The DNA samples were subjected toagarose electrophoresis. The ethidium bromide-stained images are shownin FIG. 11.

As can be seen in FIG. 11 that, in culture at 37° C., whereas no DNAfragment was detected in 1-2-3 cells (lane 1), DNA fragmentation to 180bp oligomers could be detected in the p51A gene-transfected 1C1 and 4B1cells (lanes 2 and 3).

In culture at 32° C., DNA fragmentation was detected in 1-2-3 cells(lane 4) and the DNA fragmentation was promoted in 1C1 cells and 4B1cells (lanes 5 and 6). These results were consonant with the results ofmorphological observation of apoptosis and the results of the growthinhibition test of p51-introduced cells (32° C., 37° C.).

The presence or absence of apoptotic morphological changes in cells wasstudied by fixing the respective cells on glass slides and, after Gimsastaining, observing the cell morphology and the degree of stainingmicroscopically. The viable count of cells was found by Trypan Bluestaining and counting.

As a result, the cells grown at 32° C. had surface projections andpresented with a shrunken, strained or constricted form. Moreover, inGimsa-stained cell specimens, chromatin condensation was observed eitheraround the nuclear envelope or in intracellular masses. In contrast, inthe cells cultured at 37° C., no such morphological change was observed.

Within 24 hours of culture at 32° C., cells undergoing apoptotic deathand cells continuing the cell cycle and growth were mixedly present, andafter 24 hours the viable cell count of p51-expressing cells was 10⁵/mland the cell count of 1-2-3 cells was 1.7×10⁵/ml.

The foregoing indicated that the p51 gene-containing cells treated atthe temperature of 32° C. experienced a sudden apoptosis in the presenceof p53. It was thus confirmed that the p51 protein, like the p53protein, induces apoptosis in a significant measure.

EXPERIMENTAL EXAMPLE 5

A specific antibody against the C-terminal region (570th˜641st positionsof the amino acid sequence) of the human p51B protein was prepared andhuman cells were immunostained.

Thus, the coding region (the 1851st˜206th positions of the nucleotidesequence) of the human p51B DNA was ligated to the GST fusion proteinexpression vector pGEX-1λT (Pharmacia) and a fusion protein wassynthesized in Escherichia coli. Using this fusion protein and BALB/Cmice, an antiserum (polyclonal antibody) was prepared in the routinemanner and absorbed with the GST protein to provide a specific antibodyagainst the C-terminal region of the p51B protein.

The above antibody was subjected to a primary reaction with a human skintissue frozen specimen and then to a secondary reaction with afluorescent-labeled goat anti-mouse IgG antibody.

As a result, this antibody specifically stained the cell nucleus fromthe spinal cell layer to the basement layer of the human skin. Thisspecificity was found because said fusion protein treatment abolishedthis reaction while the pretreatment with GST protein failed to abolishthe reaction.

INDUSTRIAL APPLICABILITY

The genes of the present invention can be characterized as related genesof the p53 gene which is known as a tumor suppressor gene. With thesegenes, the expression levels and functions in cells can be analyzed andit is expected that by analyzing their expression products, morbiditiesin various diseases associated with these genes (for example, malignanttumors) can be elucidated and diagnostic and therapeutic modalities canbe established.

Furthermore, the genes of the present invention are expressed in thegland tissues (prostate, mammary gland), muscle tissues, and thymus andother immune systems in contrast to p73 which is expressed in thenervous system and are likely to be involved in abnormalities of thesetissues. Therefore, it is expected that these genes will contribute tothe development of inhibitors or suppressants of such abnormalities.

In accordance with the present invention, there is provided a novelhuman p51 gene which is of value as a cell proliferation suppressivegene. The novel gene of the present invention resembles the gene codingfor the p53 protein or p73 protein. Therefore, the gene of the inventioncan be utilized in studies on the relationships of the analyzedfunctions of these related genes to various diseases and used in studiesfor application to gene diagnosis and the medicinal use of these genesin various diseases. Moreover, by using the gene of the invention, theexpression pattern of the gene in various human tissues can be exploredand its functions in the human body can be analyzed.

In addition, with this gene, the human p51 protein encoded by the genecan be produced in large quantities by the genetic engineeringtechnology. Thus, the gene and gene fragments provided by the presentinvention can be integrated with expression vectors to constructrecombinant human p51 proteins and study p51 protein activity and thefunctions, e.g. binding activity, of the p51 protein.

Furthermore, the p51 protein is useful for the pathological elucidation,diagnosis and therapy of diseases associated with the p51 gene or itsproduct (e.g. diseases related to the transcription activity of cellsand various diseases related to apoptosis, particularly cancers).

The p51 protein has physiological actions or functions similar to thoseof the p53 protein. Therefore, when various biological stresses such asvirus infection, cytokine stimulation, hypoxia, a change in thenucleotide pool, drug-induced metabolic derangments, etc. act upon theliving tissues, the protein of the invention exhibits such functions assignal transductions through interactions with other proteins andtranscriptional control over the other genes to thereby modulatereplication of the cell DNA in the tissues subjected to said biologicalstresses, interrupt their cell cycle to repair the cells, eliminatecells through apoptosis, or promote the differentiation of cells tothereby control to the defense of living tissues against said stresses.

In accordance with the present invention, there are provided a genetransfer vector containing the human p51 gene or an allele thereof whichis useful for gene therapy, cells into which said p51 gene or allele hasbeen introduced, a gene-therapeutic agent containing said vector orcells as an active ingredient, and a method of gene therapy exploitingthem.

Furthermore, in accordance with the present invention, there is provideda pharmaceutical product containing the p51 protein as an activeingredient which has activity to suppress growth of various cancer cellsand finds application in the treatment of various neoplastic diseasesand associated symptoms through said activity.

The functional regions of the human p51 gene and the corresponding mousegene are completely identical except for the 3 amino acids in the TAdomain, thus reflecting a high degree of conservation and suggesting theimportance of the present gene [The nucleotide sequences of the twogenes are correlately shown in FIG. 12-14 and FIG. 15].

1. An isolated protein defined under (a) or (b) below: (a) a proteincomprising the amino acid sequence of SEQ ID NO:1; or (b) a proteincomprising amino acid sequences identified by amino acids 1-59, aminoacids 142-321, and amino acids 359-397 of the amino acid sequence of SEQID NO:1 and having p51 activity.